Quantitative differential interference contrast (dic) devices for computed depth sectioning

ABSTRACT

Embodiments of the present invention relate to a method for computing depth sectioning of an object using a quantitative differential interference contrast device having a wavefront sensor with one or more structured apertures, a light detector and a transparent layer between the structured apertures and the light detector. The method comprises receiving light, by the light detector, through the one or more structured apertures. The method also measures the amplitude of an image wavefront, and measures the phase gradient in two orthogonal directions of the image wavefront based on the light. The method can then reconstruct the image wavefront using the amplitude and phase gradient. The method can then propagate the reconstructed wavefront to a first plane intersecting an object at a first depth. In one embodiment, the method propagates the reconstructed wavefront to additional planes and generates a three-dimensional image based on the propagated wavefronts.

CROSS-REFERENCES TO RELATED APPLICATIONS

This is a non-provisional application that claims benefit of the filingdate of U.S. Provisional Patent Application No. 61/205,487 entitled“Quantitative differential interference contrast (DIC) microscopy andits computed depth sectioning ability” filed on Jan. 21, 2009. Thatprovisional application is hereby incorporated by reference in itsentirety for all purposes.

This non-provisional application is related to the following co-pendingand commonly-assigned patent applications, which are hereby incorporatedby reference in their entirety for all purposes:

-   -   U.S. patent application Ser. No. 11/125,718 entitled        “Optofluidic Microscope Device” filed on May 9, 2005.    -   U.S. patent application Ser. No. 11/686,095 entitled        “Optofluidic Microscope Device” filed on Mar. 14, 2007.    -   U.S. patent application Ser. No. 11/743,581 entitled “On-chip        Microscope/Beam Profiler based on Differential Interference        Contrast and/or Surface Plasmon Assisted Interference” filed on        May 2, 2007.    -   U.S. patent application Ser. No. 12/398,098 entitled “Methods of        Using Optofluidic Microscope Devices” filed Mar. 4, 2009.    -   U.S. patent application Ser. No. 12/398,050 entitled        “Optofluidic Microscope Device with Photosensor Array” filed on        Mar. 4, 2009.    -   U.S. patent application Ser. No. 12/638,518 entitled “Techniques        for Improving Optofluidic Microscope Devices” filed on Dec. 15,        2009.    -   U.S. patent application Ser. No. 12/435,165 entitled        “Quantitative Differential Interference Contrast (DIC)        Microscopy and Photography based on Wavefront Sensors” filed May        4, 2009.

BACKGROUND OF THE INVENTION

Embodiments of the present invention generally relate to quantitativedifferential interference contrast (DIC) devices with wavefront sensors.More specifically, certain embodiments relate to quantitative DICdevices with wavefront sensors used in applications such as microscopyor photography, and that are adapted to compute depth sectioning ofspecimens and other objects.

DIC microscopes render excellent contrast for optically transparentbiological samples without the need of introducing exogenous contrastagents into the samples. Due to the noninvasive nature, DIC microscopesare widely used in biology laboratories.

Conventional DIC microscopes and other conventional DIC devicestypically operate by first creating two identical illumination lightfields exploiting polarization selection. FIG. 1 is a schematic drawingof a conventional DIC device 10 that operates by interfering slightlydisplaced duplicate image light fields of polarized light. Theconventional DIC device 10 includes an illumination source 20 providingpolarized light to an object 30. As illustrated, the light fields aretransmitted through the object 30 and are laterally displaced withrespect to each other along the x-direction. A net phase lag (typicallyπ/2) is then introduced on one of the transmitted image light fields.The two light fields are allowed to interfere with each other at theimage plane 40. More simply, the process is equivalent to duplicatingthe transmitted image light field, laterally displacing a copy slightlyand detecting the interference of the two light fields at image plane40.

Mathematically, this implies that the observed DIC intensity image 42from the conventional DIC device 10 with a magnification factor of M isgiven by Eq. (1):

$\begin{matrix}\begin{matrix}{{I_{DIC}\left( {x,y} \right)} = {{\psi_{DIC}\left( {x,y} \right)}}^{2}} \\{= {{B\left( {x,y} \right)} + {{C\left( {x,y} \right)}*{\sin \begin{pmatrix}{{\arg \left( {\psi \left( {{x - {\Delta/2}},y} \right)} \right)} -} \\{\arg \left( {\psi \left( {{x + {\Delta/2}},y} \right)} \right)}\end{pmatrix}}}}} \\{\approx {{B\left( {x,y} \right)} + {{C\left( {x,y} \right)}*\begin{pmatrix}{{\arg \left( {\psi \left( {{x - {\Delta/2}},y} \right)} \right)} -} \\{\arg \left( {\psi \left( {{x + {\Delta/2}},y} \right)} \right)}\end{pmatrix}}}}\end{matrix} & (1)\end{matrix}$

where B(x, y)=|(ψ(x−Δ/2,y))|²+|(ψ(x+Δ/2,y))|²,C(x, y)=2|(ψ(x−Δ/2,y))∥(ψ(x+Δ/2,y))|, and ψ(x, y) is the image wavefrontas relayed by the microscope for each light field, ψ_(DIC)(x, y) is theDIC image wavefront, and Δ=Ma is the relative displacement of the imagesassociated with the light fields. The last expression in Eq. (1) isvalid only in situations where the phase difference is small.

However, conventional DIC devices have several limitations. One majorlimitation is that conventional DIC devices translate phase variationsinto amplitude (intensity) variations. As shown in Eq. (1), the DICintensity image, I_(DIC)(x,y) is a sine function of the differentialphase so that the phase information cannot be interpreted directly fromthe intensity of the DIC image. Also, the B(x,y) and C(x,y) terms bothcontain amplitude information so that the DIC image contains entangledamplitude and phase information. Therefore, phase variations cannot beeasily disentangled from amplitude (intensity) variations that arisefrom absorption and/or scattering by an object. In other words,conventional DIC devices do not distinguish between the effects ofabsorption and phase variation. As a consequence of this entanglement ofamplitude and phase information and nonlinear phase gradient response,conventional DIC devices are inherently qualitative and do not providequantitative phase measurements

Another limitation of conventional DIC devices is that they usepolarized light and depend on the polarization in their phase-imagingstrategies. Since polarized light must be used, conventional DIC devicesgenerate images of birefringent objects (e.g., potato starch storagegranules) that typically suffer from significant artifacts.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention relate to methods of using aquantitative DIC device(s) to compute depth sections of an object. Anobject introduced into the quantitative DIC device alters a light fieldand induces an image wavefront having an amplitude and phase gradient.The light detector at the back of the wavefront sensor measures thedistribution of light passing through structured apertures in thewavefront sensor. The wavefront sensor uses the light distribution tomeasures separately the amplitude and phase gradient of the wavefront intwo orthogonal directions. The quantitative DIC device numericallyreconstructs the image wavefront from the amplitude and phase gradient,and computationally propagates the reconstructed wavefront to planes atdifferent depths through the thickness of the object.

One embodiment is directed to a method for computing depth sectioning ofan object using a quantitative differential interference contrast devicehaving a wavefront sensor with one or more structured apertures, a lightdetector and a transparent layer between the one or more structuredapertures and the light detector. The method comprises receiving lightby the light detector through the one or more structured apertures. Themethod further comprises measuring an amplitude of an image wavefrontbased on the received light measuring a phase gradient in two orthogonaldirections of the image wavefront based on the received light. Then, theprocessor reconstructs the image wavefront using the measured amplitudeand phase gradient and propagates the reconstructed wavefront to a firstplane intersecting an object at a first depth.

Another embodiment is directed to a wavefront sensor comprising anaperture layer having one or more structured apertures, a light detectorand a transparent layer between the aperture layer and the lightdetector. The light detector measures the amplitude of the wavefront andthe phase gradient in two orthogonal directions based on the lightreceived through the structured apertures.

Another embodiment is directed to a quantitative DIC device comprising awavefront sensor and a processor communicatively coupled to thewavefront sensor. The wavefront sensor comprises an aperture layerhaving one or more structured apertures, a light detector and atransparent layer between the aperture layer and the light detector. Thelight detector measures the amplitude of the wavefront and the phasegradient in two orthogonal directions based on the light receivedthrough the structured apertures. The processor reconstructs thewavefront using the measured amplitude and phase gradient.

These and other embodiments of the invention are described in furtherdetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a schematic drawing of a conventional DIC device.

FIG. 2 is schematic drawing of a quantitative DIC device, according toan embodiment of the invention.

FIG. 3 is schematic drawing of a side view of components of aquantitative DIC device in a first configuration, according to anembodiment of the invention.

FIG. 3 is schematic drawing of a side view of components of aquantitative DIC device in a first configuration, according to anembodiment of the invention.

FIG. 4 is a schematic drawing of a side view of components of aquantitative DIC device in a second configuration, according to anembodiment of the invention.

FIG. 5 is a schematic drawing of a side view of components of aquantitative DIC device in a third configuration, according to anembodiment of the invention.

FIG. 6( a) is a schematic drawing of a side view of components of aquantitative DIC device having a SAI wavefront sensor, according to anembodiment of the invention.

FIG. 6( b) is a schematic drawing of a side view of components of aquantitative DIC device having a SAI wavefront sensor, according to anembodiment of the invention.

FIG. 6( c) is a schematic drawing of a side view of components of aquantitative DIC device having a SAI wavefront sensor, according to anembodiment of the invention.

FIG. 7( a)(1) is a schematic drawing of a perspective view of atwo-dimensional structured aperture in the form of a ‘plus’ signconfiguration, according to an embodiment of the invention.

FIG. 7( a)(2) is a schematic drawing of a perspective view of atwo-dimensional structured aperture in the form of a ‘plus’ signconfiguration, according to an embodiment of the invention.

FIGS. 7( b), 7(c), and 7(d) are images taken by a scanning electronmicroscope of two-dimensional structured apertures, according toembodiments of the invention.

FIG. 8 is a schematic drawing of a side view of components of aquantitative DIC device having a SAI wavefront sensor, in accordancewith embodiments of the invention.

FIG. 9 is a schematic drawing of a side view of components of aquantitative DIC device having a Shack-Hartmann wavefront sensor, inaccordance with an embodiment of the invention.

FIG. 10( a) is a schematic drawing of a top view of components of anintensity OFM device including light transmissive regions in the form ofa one-dimensional array of single light transmissive regions.

FIG. 10( b) is a schematic drawing of a top view of components of aquantitative DIC device having an OFM wavefront sensor, according to anembodiment of the invention.

FIG. 11 illustrates a schematic diagram illustrating this propagationapproach, according to embodiments of the invention.

FIG. 12 is a schematic diagram of the propagation approach using aprocessor, according to embodiments of the invention.

FIGS. 13( a) and 12(b) are schematic drawings of a focusing approachtaken by a traditional microscope.

FIG. 14 is a flow chart of a compute depth sectioning method using aquantitative DIC device having a wavefront sensor, according toembodiments of the invention.

FIG. 15( a) is a side view of components of the wavefront sensor,according to an embodiment of the invention.

FIG. 15( b) is a top view of components of the wavefront sensor in FIG.15( a), according to an embodiment of the invention.

FIG. 15( c) is a sectional view of components of the wavefront sensor inFIG. 15( a) through the center of structured aperture, according to anembodiment of the invention.

FIG. 16( a) is an intensity/amplitude image taken of a starfish embryousing a quantitative DIC microscope having an SAI wavefront sensor,according to an embodiment of the invention.

FIG. 16( b) is an image based on phase gradient in the x direction takenof a starfish embryo using a quantitative DIC device having an SAIwavefront sensor, according to an embodiment of the invention.

FIG. 16( c) is an image based on phase gradient in the y direction takenof a starfish embryo using a quantitative DIC device having an SAIwavefront sensor, according to an embodiment of the invention.

FIGS. 16( d), 16(e) and 16(f) showcase some of the unwrapping algorithmswhen applied to the raw amplitude, differential phase x and differentialphase y data of FIGS. 16( a), 16(b) and 16(c), according to embodimentsof the invention.

FIG. 17( a) is an image of potato starch storage granules in immersionoil taken by a conventional transmission microscope.

FIG. 17( b) is an image of potato starch storage granules in immersionoil taken by a conventional DIC microscope.

FIG. 17( c) is an intensity image of potato starch storage granules inimmersion oil taken by a quantitative DIC device in a microscope system,according to an embodiment of the invention.

FIG. 17( d) is an artifact-free x-direction phase image of potato starchstorage granules in immersion oil taken by a quantitative DIC device ina microscope system, according to an embodiment of the invention.

FIG. 17( e) is an artifact-free y-direction phase image of potato starchstorage granules in immersion oil taken by a quantitative DIC device ina microscope system, according to an embodiment of the invention.

FIG. 18 shows a block diagram of subsystems that may be present incomputer devices that are used in quantitative DIC device, according toembodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will be described below withreference to the accompanying drawings. Some embodiments include asimple and quantitative DIC device with a wavefront sensor that can beused in applications such as microscopy, photography, or other imagingapplications.

Wavefront sensors of embodiments of the invention can be in any suitableform. For example, the wavefront sensor can be in the form of a singlepixel (element) wavefront sensor. In another example, the wavefrontsensor can be in the form of a one dimensional wavefront sensor arrayhaving sensor elements (e.g., pixels) located along a single direction.In another example, the wavefront sensor can be in the form of atwo-dimensional wavefront sensor array comprising sensor elementslocated along two orthogonal directions.

In general, quantitative DIC devices of embodiments of the inventionprovide advantages because they can separately measure the amplitude andphase gradient of an image wavefront in two orthogonal directions. Withthis information, the quantitative DIC device has sufficient data tonumerically reconstruct an image wavefront and propagate it to otherplanes.

Quantitative DIC devices of embodiments of the invention also provideadvantages because they do not require polarized light as part of theirimaging technique. Since these quantitative DIC devices are notdependent on the polarization of the light (illumination), these devicescan use unpolarized light to generate artifact-free DIC images for bothbirefringent and homogenous objects. Also, an ordinary light source canbe used such as the light source used in a conventional microscope.Another advantage of the quantitative DIC devices of embodiments of theinvention is that they integrate DIC functionality onto a simplewavefront sensor. This integration is advantageous over conventional DICdevices which use bulky optical elements to provide DIC functionality.For this reason, embodiments of the present invention are more compact,less expensive, and simpler in use and design than conventional DICdevices.

Quantitative DIC devices of embodiments of the invention operate byselectively combining and interfering light fields of unpolarized lightat two adjacent points as illustrated in FIG. 2. FIG. 2 is schematicdrawing of a quantitative DIC device 100, according to an embodiment ofthe invention. The quantitative DIC device 100 includes an illuminationsource 20 providing unpolarized light to an object 30. In thisillustrated embodiment, the quantitative DIC device 100 employs a phasecomparison that selectively combines and interferes the light fields ofunpolarized light at two adjacent points of the image with a separationa. The quantitative DIC device 100 uses the phase comparison to generatethe image 42 of the object 30 at the image plane 40. Quantitative DICdevices employing this phase comparison can separately measure theamplitude and phase gradient of the light scattered by, or transmittedthrough, the object 30.

I. Quantitative DIC Device Configurations

Three configurations of quantitative DIC devices are described below.The first configuration includes a quantitative DIC device with a singlepixel wavefront sensor. In this configuration, raster scanning can beemployed to measure two-dimensional data about the image wavefront. Thesecond configuration includes a two-dimensional wavefront sensor arraywhich can measure the two-dimensional data about the image wavefront atthe same time, without the need for raster scanning. The first andsecond configurations use a wavefront relay system to project the imagewavefront from the object to the wavefront sensor. The thirdconfiguration eliminates the wavefront relay system of the previous twoconfigurations.

In some embodiments, the quantitative DIC devices of theseconfigurations do not depend on the polarization of light as part oftheir imaging method. These quantitative DIC devices can generateartifact-free DIC images, even for birefringence objects, if unpolarizedlight is used.

A. First Configuration

FIG. 3 is a schematic drawing of a side view of components of aquantitative DIC device 100 in a first configuration, according to anembodiment of the invention. In this configuration, the quantitative DICdevice 100 includes a single pixel wavefront sensor 110. An illuminationsource 20 provides light to an object 30 being imaged by thequantitative DIC device 100. The object 30 modulates or otherwise altersthe light and induces an image wavefront 120. The quantitative DICdevice 100 also includes a wavefront relay system 130 (e.g., one or morelenses) in communication with the single pixel wavefront sensor 110. Thewavefront relay system 130 projects or otherwise relays the imagewavefront 120 generated by the object 30 onto the single pixel wavefrontsensor 110. The single pixel wavefront sensor 110 measures the localintensity and/or slope of the projected image wavefront 120 induced by apoint of the object 30, which conjugates with the single pixel wavefrontsensor 110.

The quantitative DIC device 100 further includes a raster scanningdevice 140 for scanning the object 30 or scanning the single pixelwavefront sensor 110 to generate two-dimensional maps of the localintensity and/or slope of the image wavefront 120 induced by the object30. The quantitative DIC device 100 further includes a host computer 150having a processor 152 in communication with a computer readable medium(CRM) 154. The host computer 150 is in communication with the singlepixel wavefront sensor 110 to receive wavefront data. One or morecomponents of the quantitative DIC device 100 can be located within abody, which can be a multi-layer structure or a single, monolithicstructure.

The quantitative DIC device 100 measures the two-dimensional amplitudeand measures the phase gradient in two orthogonal directions of theimage wavefront 120 based on the measured intensity distribution. Thetotal transmission of the interference is proportional to the averageimage intensity at the aperture plane. The offsets (Δs and Δt) of thezero-order interference spot are related to the wavelength-normalizedphase gradients (θ_(x) and θ_(y)) at the aperture, respectively, throughthe spacer (transparent layer) thickness (H) and refractive index (n)as:

$\begin{matrix}{{\theta_{x}\left( {x,y} \right)} = {{\frac{\lambda}{2\pi}\frac{\partial{\phi \left( {x,y} \right)}}{\partial x}} \approx {n\; \frac{\Delta \; s}{D}}}} & (2) \\{{\theta_{y}\left( {x,y} \right)} = {{\frac{\lambda}{2\pi}\frac{\partial{\phi \left( {x,y} \right)}}{\partial x}} \approx {n\; {\frac{\Delta \; t}{D}.}}}} & (3)\end{matrix}$

Using an unwrapping method, the quantitative DIC device 100 cannumerically reconstruct the two-dimensional image wavefront 120associated with the object 30 using the measured two-dimensionalamplitude and phase gradient information. The quantitative DIC device100 can then computationally propagate the image wavefront 120 to one ormore z-planes at different depths through the thickness of the object30. The quantitative DIC device 100 can generate an intensity image, aphase gradient image in the x-direction, and a phase gradient image inthe y-direction of the object 30, a reconstructed image, and propagatedtwo dimensional images at different depths through the thickness of theobject 30. In some cases, the two dimensional images are cross-sectionalimages of the object 30. The quantitative DIC device 100 can alsocombine the two-dimensional wavefront data to generate three-dimensionaldata and images of the object 30. The computing depth sectioning methodwill be described in further detail in following sections.

An illumination source 20 can refer to any suitable device or othersource of light such as ambient light. The light provided byillumination source 20 can be of any suitable wavelength and intensity.Also, the light can include polarized and/or unpolarized light. Inembodiments where unpolarized light is used, the quantitative DIC device100 can generate artifact-free DIC images of birefringence specimens orother objects 30 in samples. Suitable illumination sources 20 arenaturally and commercially available. In some embodiments, theillumination source 20 can be a component of the quantitative DIC device100. In other embodiments, the illumination source can be a separatefrom the quantitative DIC device 100.

An illumination source 20 can be placed in any suitable location andpositioned in any suitable direction to provide appropriate light to theobject 30. In some embodiments, multiple illumination sources 20 providelight in one or more directions. For example, a camera system includinga quantitative DIC device may have a first illumination source 20 thatprovides light in a direction from the wavefront sensor to the object 30such as from a flash and a second illumination source 20 that provideslight in another direction. In other embodiments, a single illuminationsource provides light in a single direction. For example, a microscopesystem comprising a quantitative DIC device 100 may have a singleillumination source 20 positioned to provide light in the negativez-direction.

A wavefront relay system 130 can refer to a device or combination ofdevices configured to relay (e.g., project) the image wavefront 120induced by the object 30 onto a wavefront sensor such as the singlepixel wavefront sensor 110 in FIG. 3 or the wavefront sensor array 210shown in FIGS. 4 and 5. In one example, a wavefront relay system 130includes one or more lenses. The light may be relayed in any suitablemanner. In FIG. 3, the wavefront relay system 130 projects the imagewavefront 120 from the object 30 onto a single pixel wavefront sensor110.

A raster scanning device 140 can refer to any suitable device for rasterscanning the object 30 or raster scanning the wavefront sensor. Theraster scanning device 140 can be a component of the quantitative DICdevice 100 in some embodiments and can be a separate device in otherembodiments.

In the illustrated embodiment, the host computer 150 is a component ofthe quantitative DIC device 100. In other embodiments, the host computer150 can be a separate device. Although the processor 152 and CRM 154 areshown as components of the quantitative DIC device 100, in otherembodiments the processor 152 and/or CRM 154 can be components of thewavefront sensor.

A processor (e.g., a microprocessor) can refer to any suitable devicefor processing the functions of the quantitative DIC device 100. In theillustrated embodiment, the processor 150 receives signals withwavefront data associated with the intensity distribution and/or slopedata of the image wavefront 120 measured by the single pixel wavefrontsensor 110. The wavefront data may include a two-dimensional map of theintensity distribution and/or slope of the image wavefront 120 measuredby the single pixel wavefront sensor 110 by employing the rasterscanning device 140, the amplitude and phase gradient data associatedwith the image wavefront 120, the wavelength(s) of the light, and/orother information about the light received by the single pixel wavefrontsensor 110. The processor 150 executes code stored on the CRM 154 toperform some of the functions of the quantitative DIC device 100. Thesefunctions include interpreting the light distribution and/or slope datameasured by the single pixel wavefront sensor 110, measuring the centerof a projection through a structured aperture, separating theprojections from structured apertures, determining offsets of theprojections or the focal points, determining the amplitude and phasegradient of the image wavefront 120 in two orthogonal directions usingthe light distribution data, reconstructing the image wavefront usingthe amplitude and phase gradient data in two orthogonal directions,propagating the reconstructed wavefront from the detector z plane to oneor more z planes, generating one or more two-dimensional images based onintensity, phase gradient in the x-direction, phase gradient in they-direction, the reconstructed wavefront, and/or the propagatedwavefronts, combining two-dimensional image data to generatethree-dimensional data and images of the object 30, displaying one ormore images of the object 30, and other functions associated withcomputed depth sectioning and image processing.

A CRM 154 can refer to a memory that stores data and may be in anysuitable form including a memory chip, etc. The CRM 154 stores the codefor performing some functions of the quantitative DIC device 100. Thecode is executable by the processor 152. In one embodiment, the CRM 154comprises a) code for interpreting the light distribution data receivedfrom the single pixel wavefront sensor 110, b) code for generating localslope data from the light distribution data, c) code for determining theamplitude of the image wavefront 120, and determining the phase gradientof the image wavefront 120 in two orthogonal directions using the lightdistribution data, d) code for reconstructing the image wavefront usingthe amplitude data and the phase gradient data in two orthogonaldirections, e) code for propagating the reconstructed wavefront from thedetector z plane to one or more z planes, f) code for generating one ormore two-dimensional images based on intensity, phase gradient in thex-direction, phase gradient in the y-direction, the reconstructedwavefront, and/or the propagated wavefronts, and g) code for combiningtwo-dimensional image data to generate three-dimensional data and imagesof the object 30, h) code for displaying one or more images of theobject 30, and i) any other suitable code for computed depth sectioningand image processing. The CRM 154 may also include code for performingany of the signal processing or other software-related functions thatmay be created by those of ordinary skill in the art. The code may be inany suitable programming language including C, C++, Pascal, etc.

Although not shown, the quantitative DIC device 100 may also include adisplay communicatively coupled to the processor 152. Any suitabledisplay may be used. In one embodiment, the display may be a part of thequantitative DIC device 100. The display may provide information such asthe image of the object 30 to a user of the quantitative DIC device 100.In addition, the quantitative DIC device 100 may also have an inputdevice communicatively coupled to the processor 152.

B. Second Configuration

FIG. 4 is a schematic drawing of a side view of components of aquantitative DIC device 100 in a second configuration, according to anembodiment of the invention. The second configuration of thequantitative DIC device 100 includes a two-dimensional wavefront sensorarray 210 of sensor elements for measuring two-dimensional data aboutthe image wavefront 120 in a single snapshot reading without the need ofraster scanning.

In the illustrated example, the quantitative DIC device 100 includes anillumination source 20 for providing light to an object 30 being imaged.The object 30 modulates or otherwise alters the light and induces animage wavefront 120. Although a single illumination source 20 providinglight in a single direction is shown in the illustrated embodiment,multiple illumination sources can be used providing light in one or moredirections.

The quantitative DIC device 100 also includes a wavefront relay system130 in communication with the wavefront sensor array 210. The wavefrontrelay system 130 projects or otherwise relays the image wavefront 120generated by the object 30 onto the wavefront sensor array 210. Eachsensor element (pixel) of the wavefront sensor array 210 measures thelocal intensity and slope of the image wavefront 120 induced by a pointof the object 30 which conjugates with the sensor element (pixel). Inthis case, the quantitative DIC device 100 naturally measurestwo-dimensional maps of the local intensity and slope of the imagewavefront 120 modulated by the object 30 at the same time. Although araster scanning device is not shown in the illustrated embodiment,another embodiment can include a raster scanning device 140 to rasterscan the object 30 or the wavefront sensor array 210 to form moredensely sampled images.

The quantitative DIC device 100 also includes a host computer 150 havinga processor 152 in communication with a computer readable medium (CRM)154. The host computer 150 is in communication with the wavefront sensorarray 210 to receive wavefront data. One or more components of thequantitative DIC device 100 can be located within a body, which can be amulti-layer structure or a single, monolithic structure.

The quantitative DIC device 100 measures the two-dimensional amplitudeand phase gradient of the image wavefront 120 based on the measuredintensity distribution using Eqs. (2) and (3). Using an unwrappingmethod, the quantitative DIC device 100 can reconstruct thetwo-dimensional image wavefront 120 associated with the object 30 usingthe measured two-dimensional amplitude and phase gradient information.The quantitative DIC device 100 can computationally propagate thereconstructed image wavefront 120 to one or more z-planes at differentdepths through the thickness of the object 30. The quantitative DICdevice 100 can generate an intensity image, a phase gradient image inthe x-direction, and a phase gradient image in the y-direction of theobject 30, a reconstructed image, and propagated two dimensional imagesat different depths through the thickness of the object 30. In somecases, the two dimensional images are cross-sectional images of theobject 30. The quantitative DIC device 100 can also combine thetwo-dimensional wavefront data to generate three-dimensional data andimages of the object 30. The computing depth sectioning method will bedescribed in further detail in following sections.

In the illustrated embodiment, the host computer 150 is a component ofthe quantitative DIC device 100. In other embodiments, the host computer150 can be a separate device. Although the processor 152 and CRM 154 areshown as components of the quantitative DIC device 100, in otherembodiments the processor 152 and/or CRM 154 can be components of thewavefront sensor.

In the illustrated embodiment, the processor 150 receives signals withwavefront data associated with the intensity distribution and slope ofthe image wavefront 120 measured by the wavefront sensor array 210. Thewavefront data may include the two-dimensional map of the intensitydistribution and slope of the image wavefront 120 measured by thewavefront sensor array 210, the amplitude and phase gradient informationassociated with the image wavefront 120, the wavelength(s) of the light,and/or other information about the light received by the wavefrontsensor array 210. The processor 150 executes code stored on the CRM 154to perform some of the functions of the quantitative DIC device 100 suchas interpreting the intensity distribution and/or slope data measured bywavefront sensor array 210, generating amplitude and phase gradientinformation associated with the image wavefront 120 induced by theobject 30, reconstructing an image wavefront 120 using the amplitude andphase gradient information, computing depth sectioning of the object bynumerically propagating the image wavefront 120 back to multiplez-planes through the depth of the object 30 to generate two-dimensionalimage information about the object 30, and generating three-dimensionalinformation about the object 30 from the two-dimensional informationmultiple z-planes of the object 30.

A CRM 154 can refer to any computer readable medium (e.g., memory) thatstores data and may be in any suitable form including a memory chip,etc. The CRM 154 stores the code for performing some functions of thequantitative DIC device 100. The code is executable by the processor. Inone embodiment, the CRM 154 includes a) code for interpreting the lightdistribution data received from the wavefront sensor array 210, b) codefor generating local slope data from the light distribution data, c)code for determining the amplitude of the image wavefront 120, anddetermining the phase gradient of the image wavefront 120 in twoorthogonal directions using the light distribution data, c) code forreconstructing the image wavefront using the amplitude and phasegradient data in two orthogonal directions, d) code for propagating thereconstructed wavefront from the detector z plane to one or more zplanes, e) code for generating one or more two-dimensional images basedon intensity, phase gradient in the x-direction, phase gradient in they-direction, the reconstructed wavefront, and/or the propagatedwavefronts, and f) code for combining two-dimensional image data togenerate three-dimensional data and images of the object 30, g) code fordisplaying one or more images of the object 30, and h) any othersuitable code for computed depth sectioning and image processing. TheCRM 154 may also include code for performing any of the signalprocessing or other software-related functions that may be created bythose of ordinary skill in the art. The code may be in any suitableprogramming language including C, C++, Pascal, etc.

Although not shown, the quantitative DIC device 100 may also include adisplay communicatively coupled to the processor 152. Any suitabledisplay may be used. In one embodiment, the display may be a part of thequantitative DIC device 100. The display may provide information such asthe image of the object 30 to a user of the quantitative DIC device 100.

C. Third Configuration

FIG. 5 is a schematic drawing of a side view of components of aquantitative DIC device 100 in a third configuration, according to anembodiment of the invention. The third configuration of the quantitativeDIC device 100 eliminates the wavefront relay system 130 included in thefirst and second configurations. In the illustrated embodiment, thequantitative DIC 100 includes a two-dimensional wavefront sensor array210 of sensor elements for measuring two-dimensional data about theimage wavefront 120 at the same time. In another embodiment, aquantitative DIC 100 in the third configuration comprises a single pixelwavefront sensor 110 and a raster scanning device 140. In yet anotherembodiment, a quantitative DIC 100 in the third configuration comprisesa one dimensional OFM wavefront sensor array that uses an optofluidicmicroscope (OFM) scanning scheme shown in FIGS. 10( a) and 10(b). Inthis embodiment, the sensor elements detect time varying data as theobject 30 passes through a fluid channel in the form of line scans. Theline scans are compiled to generate two-dimensional data.

In the illustrated example shown in FIG. 5, the quantitative DIC device100 includes an illumination source 20 providing light to an object 30being imaged. The object 30 modulates or otherwise alters the light,which induces the image wavefront 120. Although a single illuminationsource 20 providing light in a single direction is shown in theillustrated embodiment, multiple illumination sources can be usedproviding light in one or more directions.

The quantitative DIC device 100 also includes a host computer 150 havinga processor 152 in communication with a computer readable medium (CRM)154. The host computer 150 is in communication with the wavefront sensorarray 210 to receive wavefront data.

Each sensor element (pixel) of the wavefront sensor array 210 measuresthe local intensity and slope of the image wavefront 120 induced by apoint of the object 30 which conjugates with the sensor element (pixel).In this case, the quantitative DIC device 100 naturally measurestwo-dimensional maps of the local intensity and slope of the imagewavefront 120 modulated by the object 30 at the same time. Although araster scanning device is not shown in the illustrated embodiment,another embodiment can include a raster scanning device 140 to rasterscan the object 30 or the wavefront sensor array 210 to form moredensely sampled images.

The quantitative DIC device 100 measures the two-dimensional amplitudeand phase gradient of the image wavefront 120 based on the measuredintensity distribution using Eqs. (2) and (3). Using an unwrappingmethod, the quantitative DIC device 100 can reconstruct thetwo-dimensional image wavefront 120 associated with the object 30 usingthe measured two-dimensional amplitude and phase gradient information.The quantitative DIC device 100 can computationally propagate thereconstructed image wavefront 120 to one or more z-planes at differentdepths through the thickness of the object 30. The quantitative DICdevice 100 can generate an intensity image, a phase gradient image inthe x-direction, and a phase gradient image in the y-direction of theobject 30, a reconstructed image, and propagated two dimensional imagesat different depths through the thickness of the object 30. In somecases, the two dimensional images are cross-sectional images of theobject 30. The quantitative DIC device 100 can also combine thetwo-dimensional wavefront data to generate three-dimensional data andimages of the object 30. The computing depth sectioning method will bedescribed in further detail in following sections.

In the illustrated embodiment, the host computer 150 is a component ofthe quantitative DIC device 100. In other embodiments, the host computer150 can be a separate device. Although the processor 152 and CRM 154 areshown as components of the quantitative DIC device 100, in otherembodiments the processor 152 and/or CRM 154 can be components of thewavefront sensor.

In the illustrated embodiment, the processor 150 receives signals withwavefront data associated with the intensity distribution and slope ofthe image wavefront 120 measured by the wavefront sensor array 210. Thewavefront data may include the two-dimensional map of the intensitydistribution and slope of the image wavefront 120 measured by thewavefront sensor array 210, the amplitude and phase gradient informationassociated with the image wavefront 120, the wavelength(s) of the light,and/or other information about the light received by the wavefrontsensor array 210. In some embodiments, the wavefront data furtherincludes time varying information from an OFM wavefront sensor which canbe in the form of line scans. The processor 150 executes code stored onthe CRM 154 to perform some of the functions of the quantitative DICdevice 100 such as interpreting the intensity distribution and/or slopedata measured by wavefront sensor array 210, generating amplitude andphase gradient information associated with the image wavefront 120induced by the object 30, reconstructing an image wavefront 120 usingthe amplitude and phase gradient information, computing depth sectioningof the object by numerically propagating the image wavefront 120 back tomultiple z-planes through the depth of the object 30 to generatetwo-dimensional image information about the object 30, and generatingthree-dimensional information about the object 30 from thetwo-dimensional information multiple z-planes of the object 30.

A CRM 154 can refer to any computer readable medium (e.g., memory) thatstores data and may be in any suitable form including a memory chip,etc. The CRM 154 stores the code for performing some functions of thequantitative DIC device 100. The code is executable by the processor. Inone embodiment, the CRM 154 comprises a) code for interpreting the lightdistribution data received from the wavefront sensor array 210, b) codefor generating local slope data from the light distribution data, c)code for determining the amplitude of the image wavefront 120, anddetermining the phase gradient of the image wavefront 120 in twoorthogonal directions using the light distribution data, d) code forreconstructing the image wavefront using the amplitude of the imagewavefront 120, and determining the phase gradient data in two orthogonaldirections, e) code for propagating the reconstructed wavefront from thedetector z plane to one or more z planes, f) code for generating one ormore two-dimensional images based on intensity, phase gradient in thex-direction, phase gradient in the y-direction, the reconstructedwavefront, and/or the propagated wavefronts, and g) code for combiningtwo-dimensional image data to generate three-dimensional data and imagesof the object 30, h) code for displaying one or more images of theobject 30, and i) any other suitable code for computed depth sectioningand image processing. The CRM 154 may also include code for performingany of the signal processing or other software-related functions thatmay be created by those of ordinary skill in the art. The code may be inany suitable programming language including C, C++, Pascal, etc.

Although not shown, the quantitative DIC device 100 may also include adisplay communicatively coupled to the processor 152. Any suitabledisplay may be used. In one embodiment, the display may be a part of thequantitative DIC device 100. The display may provide information such asthe image of the object 30 to a user of the quantitative DIC device 100.

In addition, the quantitative DIC device 100 of some embodiments mayalso include a body which incorporates one or more components of thequantitative DIC device 100. The body can be a multi-layer structure ora single, monolithic structure.

In an embodiment where the quantitative DIC device 100 includes an OFMwavefront sensor, the body is a multi-layer structure. The body forms orincludes a fluid channel having a first surface. The body also includesan opaque or semi-opaque aperture layer that is an inner surface layerof the fluid channel. The opaque or semi-opaque aperture layer has lighttransmissive regions 222 in it. The opaque or semi-opaque aperture layercan be a thin metallic layer in some cases. The body may optionallyinclude a transparent protective layer (not shown) that covers theopaque or semi-opaque aperture layer to isolate the opaque orsemi-opaque aperture layer from the fluid and the object 30 movingthrough the fluid channel.

In general, imaging can be done in many ways. For example, objectscanning can be replaced by wavefront sensor scanning by the rasterscanning device. As another example, the two-dimensional raster scanningcan be replaced by one-dimensional (1D) optofluidic microscope (OFM)scanning scheme described in U.S. patent application Ser. Nos.11/686,095, 11/743,581, 12/638,518, which are hereby incorporated byreference in their entirety for all purposes. An embodiment of aquantitative DIC device that uses a one dimensional OFM scanning schemeis also described in detail in sections below.

The quantitative DIC devices of embodiments of the invention can beimplemented in various ways. In one embodiment, a quantitative DICdevice can be in the form of a wavefront sensing chip for use with amicroscope, camera or other imaging device. The wavefront sensing chipcan be communicatively coupled to the imaging device through a port inthe imaging device or the chip can be placed in a housing of the imagingdevice that accepts the chip. By coupling the wavefront sensing chip tothe imaging device, the device can be provided with quantitative DICfunctionality such as the ability to capture phase gradient images andcompute depth sectioning. For example, a quantitative DIC device can beutilized as a wavefront sensing component of an adaptive optics device.The adaptive optics device operates by measuring distortions in thewavefront and compensating for them with a spatial phase modulator suchas a deformable mirror or liquid crystal array.

II. Wavefront Sensor Types

Wavefront sensors of embodiments of the invention can be used in variouswavefront sensing applications including adaptive optics, opticaltesting, adaptive microscopy, retina imaging, etc. An example of usingwavefront sensors in adaptive microscopy can be found in Booth, M. J.,Neil, M. A. A., Juskaitis, R. Wilson, T, Proceedings of the NationalAcademy of Sciences of the United States of America 99, 5788 (April,2002), which is herein incorporated by reference in its entirety for allpurposes. An example of using wavefront sensors in retinal imaging canbe found in Liang, J. Z, Williams, D. R., Miller, D. T., Journal of theOptical Society of America a-Optics Image Science and Vision 14, 2884(November, 1997), which is herein incorporated by reference in itsentirety for all purposes.

The wavefront sensor, which can measure the local intensity and slope ofthe wavefront modulated by the object (sample) at the same time, can beimplemented in several different ways. Three types of wavefront sensorsare described below. The first type uses a structured apertureinterference (SAI) scheme. The second type uses a Shack-Hartmann scheme.The third type uses the optofluidic microscope (OFM) scheme. These typesof wavefront sensors can be used in either a single sensor element(pixel) configuration or in a wavefront sensor array (one or twodimensions) of sensor elements. If a single pixel wavefront sensor isused, a raster scanning device can be used to scan the object or thewavefront sensor to measure two-dimensional image data. If atwo-dimensional array of wavefront sensors is used, the array cancapture the two-dimensional image in a single snapshot reading. Althoughthree types of wavefront sensors are described below, any suitable typeof wavefront sensor can be used in embodiments of quantitative DICdevices.

Wavefront sensors of embodiments can be implemented in conjunction withan imaging system (e.g., a camera system, microscope system, etc.) toprovide capabilities such as computing depth sectioning of an object. Inthese implementations, one or more wavefront sensors can be coupled tothe imaging device (e.g., a microscope or camera) or inserted within animaging device to provide the additional capabilities.

A. Structured Aperture Interference (SAI) Wavefront Sensor

The basic concept of the SAI wavefront sensor is similar to the Young'sdouble-slit phase decoding scheme described in U.S. patent applicationSer. Nos. 11/743,581, 11/686,095, and 12/638,518 which is herebyincorporated by reference in its entirety for all purposes. Generally,the local slope of the image wavefront can be measured by looking at theoffset of the zero diffraction order on the back detector array (lightdetector) and the local intensity (amplitude) of the image wavefront canbe measured by integrating the diffraction signal on the back detectorarray.

FIGS. 6( a), 6(b) and 6(c) are schematic drawings of a side view ofcomponents of a quantitative DIC device 100 having a SAI wavefrontsensor, according to embodiments of the invention. The SAI wavefrontsensor can be a single pixel wavefront sensor 110 or a wavefront sensorarray 210 of one or two dimensions. In the illustrated examples, the SAIwavefront sensor 110/210 is a single pixel wavefront sensor 110 or canbe a component of a wavefront sensor array 210 of one or two dimensions.

The SAI wavefront sensor 110/210 comprises an aperture layer 220 (e.g.,a metal film), a light detector 230 and a transparent layer 240 betweenthe aperture layer 220 and the light detector 230. The aperture layer230 includes a first light transmissive region 222(a) and a second lighttransmissive region 222(b). The two light transmissive regions 222(a)and 222(b) are located at a distance D away from the light detector 230and are separated from each other by a spacing Δx. The transparent layer240 between the light detector 230 and the aperture layer 220 caninclude one or more layers of transparent material such as water or aviscous polymer (e.g., SU-8 resin), or can be a vacuum or gas-filledspace. An illumination source 20 provides illumination (light) to anobject 30 which modulates or otherwise alters the light inducing animage wavefront. The light detector 230 measures the distribution oflight received from the light transmissive regions 222(a) and 222(b).

A light detector 230 (e.g., photosensor) can refer to any suitabledevice capable of detecting light and generating signals with wavefrontdata about the intensity, wavelength, wavefront slope, phase gradient inone or more orthogonal directions, and/or other information about thelight being detected. The signals may be in the form of electricalcurrent that results from the photoelectric effect. Some examples ofsuitable light detectors 230 include a charge coupled device (CCD) or alinear or two-dimensional array of photodiodes (e.g., avalanchephotodiodes (APDs)). A light detector 230 could also be a complementarymetal-oxide-semiconductor (CMOS) or photomultiplier tubes (PMTs). Othersuitable light detectors 230 are commercially available.

The light detector 230 comprises one or more light detecting elements232. The light detecting elements 232 can be of any suitable size (e.g.,1-4 microns) and any suitable shape (e.g., circular or square). Thelight detecting elements 232 can be arranged in any suitable form suchas a one-dimensional array, a two-dimensional array, and a multiplicityof one-dimensional and/or two-dimensional arrays. The arrays can haveany suitable orientation or combination of orientations. In some cases,the light detecting elements 232 can be arranged in the same form as thelight transmissive regions 222(a) and 222(b) and correspond to the lighttransmissive regions 222(a) and 222(b). The light detector 230 alsocomprises a first surface 230(a).

In FIGS. 6( a) and 6(b), the object 30 being imaged is homogenous. InFIG. 6( c), the object 30 includes a feature 250 having a refractiveindex variation from other homogenous portions of the object 30. As anexample, the object 30 in FIG. 6( c) could be a cell and the feature 250may be a nucleus having a different refractive index than other portionsof the cell.

In FIGS. 6( a), 6(b) and 6(c), light transmissive region 222(a) collectsa reference beam of light and light transmissive region 222(b) collectsa sample beam of light. In these examples, the transparent layer(spacer) 240 has a refractive index of n and a thickness of D. When avertical plane wave is incident on the two light transmissive regions222(a) and 222(b) of the aperture layer 220, the interference pattern280 will be centered on the light detector 230. In FIG. 6( a), thevertical plane wave is incident on the two light transmissive regions222(a) and 222(b) of the aperture layer 220 and the interference pattern280(a) is centered or substantially centered on the light detector 230.In this illustrated example, the sample beam and reference beams withthe same phase exit the two light transmissive regions 222(a) and222(b), the centroid 270(a) of the light intensity distribution 280(a)of their Young's interference 260(a) is centered or substantiallycentered on the light detector 230.

If an object 30 is introduced, the phase difference induced by theobject 30 between the light transmissive regions 222(a) and 222(b) willshift the centroid 270 of the interference pattern 280 to one side. Thephase difference Δφ is directly related to the offset Δs of theinterference pattern:

$\begin{matrix}{{{\Delta \; \phi} \approx {\frac{2\pi}{\lambda}n\; \frac{\Delta \; s}{D}\Delta \; x}},} & (4)\end{matrix}$

when Δs<<D. In addition, the differential phase

$\left( \frac{\partial\phi}{\partial x} \right)$

(phase gradient) induced by the object 30 is directly related to theoffset (Δs) of the interference pattern:

$\begin{matrix}{\frac{\partial\phi}{\partial x} \approx {\frac{2\pi}{\lambda}n\; {\frac{\Delta \; s}{D}.}}} & (5)\end{matrix}$

In FIGS. 6( b) and 6(c), the reference and sample beams carry differentphases. In FIG. 6( b), the sample beam passes through a homogeneousportion of the object 30. In this case, the centroid 270(b) of the lightintensity distribution 280(b) of their Young's interference 260(b)shifts on the light detector 230 by an offset Δs₁. In FIG. 6( c), thesample beam passes through a heterogeneous portion of the object 30 andthe reference beam passes through a homogenous portion of the object 30.In this case, the centroid 270(c) of the light intensity distribution280(c) of their Young's interference 260(c) shifts on the light detector230 by an offset Δs₂. In some embodiments, the Δs₂ may be greater thanΔs₁. In other embodiments, the Δs₂ may be smaller than Δs₁.

Using Eqns. 4 and 5, the quantitative DIC device 100 can measure thephase difference and the phase gradient of the image wavefront using themeasured offset. In addition, the quantitative DIC device 100 canmeasure the local amplitude by integrating the intensity distributionmeasured by the light detector 230. By measuring the intensity and phasegradient of the light modulated by the object 30 through independentaspects of the interference pattern, the quantitative DIC device 100 canseparate the amplitude and phase information.

The one-dimensional Young's experiment with two slits can be generalizedto two dimensions by utilizing varieties of two-dimensional structuredapertures e.g., four holes, rose-shaped, ring or Fresnel zone plate, ora single hole. A two-dimensional structured aperture can refer to one ormore light transmissive regions 222 in the aperture layer of a wavefrontsensor 110/210 where the light transmissive regions extend in twoorthogonal directions (e.g., in the x- and y-directions). Withtwo-dimensional structured apertures, the SAI wavefront sensor 110/210can measure the local slope and phase gradient of the image wavefront intwo orthogonal directions (e.g., x-direction and y-direction) at thesame time. This aperture-based phase decoding scheme can be referred toas SAI wavefront sensing.

Some embodiments of quantitative DIC devices 100 have an SAI wavefrontsensor 110/210 that employs one or more two-dimensional structuredapertures to measure the amplitude and differential phase gradient intwo orthogonal directions at the same time. In these embodiments, thelight transmissive regions of the two-dimensional structure aperture areseparated by a thickness D from the light detector. The SAI wavefrontsensor 110/210 is generally located at the image plane of the imagingsystem. The structure aperture selectively transmits and combines thelight fields from two directions on the image to create an interferencepattern on the light sensor. The total transmission of the interferencepattern is proportional to the average image intensity at the lighttransmissive region.

Generally, wavefront sensors 110/210 (e.g., four holes or single holeaperture sensors) of some embodiments can measure the spatial phasegradient of the light field. Mathematically, the wavefront sensors110/210 of some embodiments measure:

G _(x)(x,y)=k _(x)(x,y)/k ₀≈(dφ(x,y)/dx)/k ₀,  (6)

G _(y)(x,y)=k _(y)(x,y)/k ₀≈(dφ(x,y)/dy)/k ₀,and  (7)

A(x,y),  (8)

where G_(x)(x, y) is the two-dimensional phase gradient in thex-direction, G_(y)(x, y) is the two-dimensional phase gradient in they-direction, and A(x, y) is the two-dimensional amplitude of thedetected wavefront. As such, a quantitative DIC device 100 with awavefront sensor 110/210 can mathematically reconstruct (unwrap) thedetected wavefront by combining the measured data appropriately. Oneunwrapping method is given by Eq. (9):

$\begin{matrix}{{\psi_{measured}\left( {x,y,z} \right)} = {{A_{measured}\left( {x,y,z} \right)}{{\exp \left( {\; {k_{0}\begin{pmatrix}{\int_{0}^{x}{G_{x}\left( {{\left( {x,{y = 0}} \right){x}} +} \right.}} \\{\int_{0}^{y}{{G_{y}\left( {{x = x},y} \right)}{y}}}\end{pmatrix}}} \right)}.}}} & (9)\end{matrix}$

Numerous approaches for reconstructing a field distribution exist(unwrapping). The unwrapping methods should all return the same answerif the signal to noise ratio (SNR) of the measurements approachesinfinity. The unwrapping methods vary in their performance based on thequantity and type of noise present in the measurements.

Embodiments of the SAI wavefront sensor 110/210 can use any suitabletype of two-dimensional structured aperture. For example, thequantitative DIC device 100 can use a two-dimensional structuredaperture in the form of a ‘plus’ sign configuration with four lighttransmissive regions (e.g., holes) arranged in orthogonal x and ydirections. An exemplary quantitative DIC device that uses a four holestructured aperture for differential phase imaging can be found in Lew,Matthew, Cui, Xiquan, Heng, Xin, Yang, Changhuei, Interference of afour-hole aperture for on-chip quantitative two-dimensional differentialphase imaging, Optic Letters, Vol. 32, No. 20, 2963 (2007), which ishereby incorporated by reference in its entirety for all purposes. Someother examples of two-dimensional structured apertures include a singlepedal-shaped aperture, a ring or Fresnel zone plate, and other suitablearrangements of light transmissive regions extending in orthogonaldirections.

In some embodiments, a quantitative DIC device 100 has a SAI wavefrontsensor 110/210 that employs a two-dimensional structured aperture offour light transmissive regions (e.g., holes) in the form of a ‘plus’sign to measure the differential phase and amplitude of an imagewavefront in the x-direction and y-direction at the same time. The twolong axes of the ‘plus’ sign are in the orthogonal x and y directionsrespectively. By placing the SAI wavefront sensor in the image plane ofan imaging system, the four transmissive regions will selectivelytransmit and combine light fields from four adjacent points on the imageto create an interference pattern read by the light detector. The totaltransmission of the interference is proportional to the average imageintensity at the light transmissive region. In addition to the spacerthickness D, the offsets offset_(x)(x, y) and offset_(y)(x, y) of thezero-order interference spot are related to the net wavefront gradientG_(x)(x, y) and G_(y)(x, y) at the light transmissive regionrespectively:

$\begin{matrix}{{{G_{x}\left( {x,y} \right)} = \frac{1}{\sqrt{1 + \left( \frac{D}{{offset}_{x}\left( {x,y} \right)} \right)^{2}}}},{{G_{y}\left( {x,y} \right)} = {\frac{1}{\sqrt{1 + \left( \frac{D}{{offset}_{y}\left( {x,y} \right)} \right)^{2}}}.}}} & (10)\end{matrix}$

FIGS. 7( a)(1) and 7(a)(2) are schematic drawings of a perspective viewof a two-dimensional structured aperture 300 in the form of a ‘plus’sign configuration, according to embodiments of the invention. In theillustrated examples, the quantitative DIC device 100 comprises anaperture layer 220 having a two-dimensional structured aperture 300 offour light transmissive regions 222 extending in both x and y directionsin a ‘plus’ sign configuration. Using the two-dimensional structuredaperture 300, the quantitative DIC device 100 can measure thedifferential phase and amplitude in both x and y directions. Thequantitative DIC device 100 also has a light detector 230 located at adistance D from the aperture layer 230. The quantitative DIC device 100also includes a transparent layer 240 with a thickness D located betweenthe aperture layer 220 and the light detector 230. The transparent layer240 can be comprised of one or more layers of transparent material suchas water or a viscous polymer (e.g., SU-8 resin) or can be a vacuum orgas-filled space. Any suitable spacing Δx between the light transmissiveregions 222 in the ‘plus’ sign configuration can be used. Some examplesof suitable spacing Δx are 1 μm, 2 μm, or 3 μm.

In FIGS. 7( a)(1) and 7(a)(2), the light detector 230 receives lightpassing through light transmissive regions 222 in two-dimensionalstructured aperture 300. In FIG. 7( a)(1), the illumination source 20projects a light field perpendicular to the first surface 220(a). InFIG. 7( a)(2), the illumination source 20 projects a light field at anangle with respect to the first surface 22(a). The light field projectedat different angles in FIG. 7( a)(1) and FIG. 7( a)(2) results indifferent projections 310(a) and 310(b) respectively onto the lightdetector 230.

FIGS. 7( b), 7(c), and 7(d) are images taken by a scanning electronmicroscope of two-dimensional structured apertures 300, according toembodiments of the invention. FIG. 7( b) illustrates a two-dimensionalstructured aperture 300 in the form of a single pedal-shaped aperture.FIG. 7( c) and FIG. 7( d) illustrate two-dimensional structuredapertures 300 of light transmissive regions 222 in the form of a ‘plus’sign configuration. The spacing between the light transmissive regions222 in the structured aperture 300 in FIG. 7( c) is shorter than thespacing between the light transmissive regions 222 in the structuredaperture 300 shown in FIG. 7( d). FIGS. 7( e), 7(f), and 7(g) are imagesof the resulting interference patterns of the two-dimensional structuredapertures 300 respectively from FIGS. 7( b), 7(c), and 7(d), accordingto embodiments of the invention. FIG. 7( h) is a schematic drawing of aFresnel-zone plate structured aperture with a circular frame.

FIG. 8 is a schematic drawing of a side view of components of aquantitative DIC device 100 having a SAI wavefront sensor 110/210, inaccordance with embodiments of the invention. The SAI wavefront sensor110/210 has an aperture layer 220 having an array 325 of threestructured apertures 300. The SAI wavefront sensor 110/210 also includesa light detector 230 and a transparent layer 240 with a thickness, Dlocated between the aperture layer 220 and the light detector 230. Thetransparent layer 240 can be comprised of one or more layers oftransparent material such as water or a viscous polymer (e.g., SU-8resin) or can be a vacuum or gas-filled space.

In the illustrated embodiment, the quantitative DIC device 100 is acomponent of a camera system with a first illumination source 20(a)providing light in the z-direction and a second illumination source20(b) (e.g., a flash) providing light from another direction. In thisexample, the light from the additional illumination source reflects offthe object 30. The object 30 modulates or otherwise alters the lightfrom both illumination sources 20(a) and 20(b) inducing an imagewavefront 120. In other embodiments, the second illumination source20(b) may be eliminated such as a quantitative DIC device 100 that is acomponent of a microscope system. In yet other embodiments, otherillumination sources may provide light from other directions.

The quantitative DIC device 100 also includes a wavefront relay system130 (e.g., one or more lenses) in communication with the wavefrontsensor 110/120. The wavefront relay system 130 relays the wavefront 120induced by the object 30 to the SAI wavefront sensor 110/120. In otherembodiments, the quantitative DIC device 100 can also include a hostcomputer 150 having a processor 152 and a computer readable medium 154.

In operation, the illumination sources 20(a) and 20(b) provide light tothe object 30 inducing the image wavefront 120. The wavefront relaysystem 130 relays the image wavefront 120 to the structured apertures300 of the SAI wavefront sensor 110/210. The light passes through thestructured apertures. The SAI wavefront sensor 110/210 measures theoffsets in the x and y directions of the zero diffraction order of thelight distribution read by the light detector 230. The SAI wavefrontsensor 110/210 measures the two dimensional phase gradient in the x andy directions based on the offsets using Eqns. (2) and (3). The SAIwavefront sensor 110/210 also measures the amplitude of the imagewavefront by integrating the intensity readings over the light detector230. The quantitative DIC device 100 can then reconstruct the imagewavefront 120 using an unwrapping method. The quantitative DIC device100 can also propagate the reconstructed wavefront to one or moreparallel planes intersecting the object 30 in order to compute depthsectioning of the object 30. The quantitative DIC device 100 can alsocompile the two-dimensional information about the reconstructed andpropagated wavefronts to generate three-dimensional information aboutthe object 30. The quantitative DIC device 100 can generate two orthree-dimensional images of the object 30 based on the amplitude, phasegradient in a first direction, and/or phase gradient in a seconddirection orthogonal to the first direction.

The quantitative DIC device 100 also includes an x-axis, a y-axis, and az-axis. The x-axis and the y-axis lie in the plane of the surface 230(a)of the light detector 230. The z-axis is orthogonal to the plane of thesurface 230(a).

B. Shack-Hartmann Wavefront Sensor

The Shack-Hartmann wavefront sensor includes a microlens array of thesame focal length. Each Shack-Hartmann wavefront sensor focuses thelocal wavefront across each microlens and forms a focal spot onto aphotosensor array. The local slope of the wavefront can then becalculated from the position of the focal spot on the light sensor. Theprinciples of the Shack-Hartmann sensors are further described in Platt,Ben C. and Shack, Roland, History and Principles of Shack-HartmannWavefront Sensing, Journal of Refractive Surgery 17, S573-S577(September/October 2001) and in Wikipedia, Shack-Hartmann, athttp://en.wikipedia.org/wiki/Shack-Hartmann (last visited Jan. 21,2009), which are which are hereby incorporated by reference in theirentirety for all purposes.

Microlenses can refer to small lenses, generally with diameters of lessthan about a millimeter and can be as small as about 10 μm. Microlensescan be of any suitable shape (e.g., circular, hexagonal, etc.).Microlenses can also have any suitable surface configuration. In oneexample, a microlens is a structure with one plane surface and onespherical convex surface to refract light. In another example, amicrolens has two flat and parallel surfaces and the focusing action isobtained by a variation of refractive index across the microlens. Inanother example, a microlens is a micro-Fresnel lens having a set ofconcentric curved surfaces which focus light by refraction. In yetanother example, a microlens is a binary-optic microlens with grooveshaving stepped edges.

A microlens array can refer to a one or two-dimensional array of one ormore microlenses. A Shack-Hartmann wavefront sensor comprises anaperture layer having a microlens array with one or more microlenseslocated within the light transmissive regions (e.g., holes) in theaperture layer. The Shack-Hartmann wavefront sensor 110/210 alsoincludes a light detector 230 and a transparent layer 240 with athickness, D located between the aperture layer 220 and the lightdetector 230. The transparent layer 240 can be comprised of one or morelayers of transparent material such as water or a viscous polymer (e.g.,SU-8 resin) or can be a vacuum or gas-filled space.

FIG. 9 is a schematic drawing of a side view of components of aquantitative DIC device 100 having a Shack-Hartmann wavefront sensor110/210, in accordance with an embodiment of the invention. In theillustrated embodiment, the Shack-Hartmann wavefront sensor 110/210 hasan aperture layer 230 comprising a microlens array 325 having threemicrolenses 330.

In the illustrated embodiment, the quantitative DIC device 100 is acomponent of a camera system with a first illumination source 20(a)providing light in the direction of the z-axis and a second illuminationsource 20(b) (e.g., a flash) providing light from another direction. Inthis example, the light from the additional illumination source reflectsoff the object 30. The object 30 alters the light from both illuminationsources 20(a) and 20(b) inducing an image wavefront 120. In otherembodiments, other illumination sources or a single illumination sourcemay be used. The quantitative DIC device 100 also includes a wavefrontrelay system 130 (e.g., one or more lenses) in communication with thewavefront sensor 110/120. In other embodiments, the quantitative DICdevice 100 can also include a host computer 150 having a processor 152and a computer readable medium 154.

In operation, the illumination sources 20 provide light to the object 30inducing the wavefront 120. The wavefront relay system 130 relays theimage wavefront 120 induced by the object 30 to the array 320 ofmicrolenses 330. Each microlens 330 concentrates light to focal spots onthe light detector 230. The Shack-Hartmann wavefront sensor 110/120measures offsets of the positions of the focal spots on the lightdetector 230. The Shack-Hartmann wavefront sensor 110/120 measures thetwo dimensional phase gradient in x-direction and y-direction and theamplitude as given by Eqns. (2) and (3). The Shack-Hartmann wavefrontsensor 110/210 also measures the amplitude of the image wavefront byintegrating the intensity readings over the light detector 230. Thequantitative DIC device 100 can then reconstruct the image wavefront 120using an unwrapping method and propagate the wavefront from the plane atthe surface 230(a) of the light detector 230 to any number of parallelplanes intersecting the object 30 to determine image data at differentdepths through the thickness of the object 30. The quantitative DICdevice 100 can also compile the two-dimensional information about thewavefront 120 to generate three-dimensional information about the object30. The quantitative DIC device 100 can generate two orthree-dimensional images of the object 30 based on the amplitude, phasegradient in a first direction, and/or phase gradient in a seconddirection orthogonal to the first direction.

The quantitative DIC device 100 also includes an x-axis, a y-axis, and az-axis. The x-axis and the y-axis lie in the plane of the surface 230(a)of the light detector 230. The z-axis is orthogonal to the plane of thesurface 230(a).

C. Optofluidic Microscope (OFM) Wavefront Sensor

The above compact and lensless two-dimensional differential phasemeasurement scheme can be deployed in OFM imaging scheme as well. Byreplacing the single light transmissive regions of an intensity OFMdevice with two-dimensional structured apertures, the intensity OFMdevice becomes an on-chip and quantitative differential interferencecontrast optofluidic microscope which can improve image quality whileproviding high throughput in a compact and inexpensive device. Thequantitative DIC device has an OFM wavefront sensor which includes anaperture layer with an array of two-dimensional structured apertures, alight detector, and a transparent layer with a thickness D between theaperture layer and the light detector. The OFM wavefront sensor candetermine the amplitude of the image wavefront of an object anddetermine the phase gradient of the image wavefront in two orthogonaldirections of the object. Young's double slit experiment provides abasis for this technique.

FIG. 10( a) is a schematic drawing of a top view of components of anintensity OFM device 400 including light transmissive regions 222 in theform of a one-dimensional array 410 of single light transmissive regions222. The intensity OFM device 400 also includes a body 420 forming orincluding a fluid channel 430. The light transmissive regions 222 arelocated in the aperture layer 440 of the body 420. The intensity OFMdevice 400 also includes a light detector 230 (shown in FIG. 4) havingelements for taking time varying readings of the light received throughthe light transmissive regions 222 as the object 30 travels through thefluid channel 430. The intensity OFM device 400 can use the time varyingreadings to reconstruct an image of the object 30 based on lightintensity detected by the light detector 230.

FIG. 10( b) is a schematic drawing of a top view of components of aquantitative DIC device 100 having an OFM wavefront sensor 210,according to an embodiment of the invention. The quantitative DIC device100 includes a body 420 comprising an OFM wavefront sensor 210 andforming or including a fluid channel 430. The body 420 can be amulti-layer structure or a single, monolithic structure. In theillustrated example, the body 420 is a multi-layer structure having anopaque or semi-opaque aperture layer 440 that is an inner surface layerof fluid channel 22. The opaque or semi-opaque aperture layer 440 haslight transmissive regions 222 in it. The opaque or semi-opaque aperturelayer 440 can be a thin metallic layer in some cases. The body 420 mayoptionally include a transparent protective layer (not shown) thatcovers the opaque or semi-opaque aperture layer 440 to isolate theopaque or semi-opaque aperture layer 440 from the fluid and the object30 moving through the fluid channel 430 of the quantitative DIC device100 having an OFM wavefront sensor 210.

The fluid channel 430 may have any suitable dimensions. For example, thewidth and/or height of the fluid channel 430 may each be less than about10, 5, or 1 micron. In some embodiments, the fluid channel 430 may besized based on the size of the objects 30 being imaged by thequantitative DIC device 100. For example, the height of the fluidchannel 430 may be 10 micron where the objects 30 being imaged are 8micron in order to keep the objects 30 close to the opaque orsemi-opaque aperture layer 440, which may help improve the quality ofthe image. In most embodiments, the flow of the fluid in the fluidchannel 430 is generally in the direction of the x-axis.

The OFM wavefront sensor 210 includes a one-dimensional array 450 ofstructured apertures 300. Each structured aperture 300 is in theconfiguration of a ‘plus’ sign configuration of light transmissiveregions 222 extending in orthogonal x and y directions. In otherembodiments, other configurations (e.g., rose-shaped, ringer shaped,single hole, etc.) can be used. In one embodiment, a microlens islocated inside one or more of the light transmissive regions forfocusing the light.

The OFM wavefront sensor 210 also includes a light detector 230 (shownin FIG. 8) having elements (e.g., pixels) for taking time varyingreadings of the light it receives from the light transmissive regions222 as the object 30 moves through the fluid channel 430. The OFMwavefront sensor 210 also includes a transparent layer (shown in FIG. 8)with a thickness, D between the aperture layer 440 and the lightdetector 230. The transparent layer 240 can be one or more layers oftransparent material such as water or a viscous polymer (e.g., SU-8resin) or can be a vacuum or gas-filled space. Any suitable spacing Δxbetween the light transmissive regions 222 in the structured apertures300 can be used. Some examples of suitable spacing Δx are 1 μm, 2 μm, or3 μm.

The quantitative DIC device 100 also includes an illumination source 20(shown in FIG. 8) to the outside of the opaque or semi-opaque aperturelayer 440. Illumination sources such as those shown in FIG. 8 canprovide light to the fluid channel 430. As a fluid flows through thefluid channel 430, an object 30 in the fluid is illuminated by theillumination source. The object 30 alters (e.g., blocks, reducesintensity, and/or modifies wavelength) the light passes through,reflecting or refracting off of it to the light transmissive regions222. The elements in the light detector 230 detect light transmittedthrough the light transmissive regions 222.

The quantitative DIC device 100 also includes an x-axis and a y-axisthat lie in the plane of the inner surface of the light detector 230proximal to the fluid channel 430. The x-axis lies along a longitudinalaxis of the fluid channel 430. The y-axis is orthogonal to the x-axis inthe plane of the inner surface of the light detector 230.

The light transmissive regions 222 in the opaque or semi-opaque aperturelayer 440 can be of any suitable shape and any suitable dimension. Inthe illustrated example, the light transmissive regions 222 are holes.The holes may be etched, for example, into the opaque or semi-opaqueaperture layer 440 (e.g., a thin metallic layer). In another embodiment,the light transmissive regions 222 may be in the form of one or moreslits. A slit can refer to an elongated opening such as a narrowrectangle. Each slit may have any suitable dimension. The slits may haveuniform dimensions or may have variable dimensions. The slits can beoriented at any suitable angle or angles with respect to the x-axis ofthe fluid channel 430.

In the illustrated embodiment, the light transmissive regions 222 in theone-dimensional array 450 of structure apertures 300 collectively extendfrom one lateral surface 430(a) to another lateral surface 430(b) of thefluid channel 430. The one-dimensional array 450 is located at an angle,θ with respect to the x-axis. The angle, θ can be any suitable angle.Although the illustrated embodiment includes a one dimensional array,other embodiments may include an OFM wavefront sensor 210 with othersuitable formation(s) of structured apertures 300 can be used such as aslit, a two-dimensional array, or a multiplicity of one-dimensionaland/or two-dimensional arrays. In addition, the formations of structuredapertures 300 can be in any suitable orientation or combination oforientations.

In operation, the light detector 230 takes time varying readings of thelight it receives from the light transmissive regions 222 as the object30 moves through the fluid channel 430. The quantitative DIC device 100uses the time varying readings to determine a two dimensional lightintensity distribution generated by ‘plus’ sign configurations of lighttransmissive regions 222. The quantitative DIC device 100 uses the lightintensity distribution to determine the interference in orthogonaldirections x and y to determine the offsets. The quantitative DIC device100 also determines the differential phase (gradient) in orthogonaldirections x and y based on the determined interference. Thequantitative DIC device 100 also determines the amplitude by summing theintensity of the light detected over an area of the light detector 230mapping to a particular set of light transmissive regions 222. Examplesof methods of measuring the amplitude and differential phase in twoorthogonal directions of the sample wavefront quantitatively can befound in Cui, Xiquan, Lew, Matthew, Yang, Changhuei, Quantitativedifferential interference contrast microscopy based onstructured-aperture interference, Appl. Phys. Lett. 93, 091113 (2008),which is hereby incorporated by reference in its entirety for allpurposes.

In the SAI and OFM wavefront sensors, structured apertures convert thephase gradient of the image wavefront into a measurable form, the offsetof the projection of the light field measured by the light detector. Ina Shack-Hartmann wavefront sensor, a microlens is used to convert thephase gradient of the image wavefront into a movement of the focal pointon the light detector. An advantage of using a SAI or OFM wavefrontsensor is that the simple structured apertures provide the ability tobuild a more compact and cost-effective wavefront sensor than theShack-Hartmann wavefront sensor having microlenses. In addition, thespacing between the structured apertures in a SAI wavefront sensor canbe much shorter than the spacing between the microlenses of theShack-Hartmann wavefront sensor. Shorter spacing can provide higherspatial resolution and denser wavefront sampling, which can beespecially beneficial when detecting complex wavefronts generated bymany biological samples.

IV. Computed Depth Sectioning

A. Approach

Computed depth sectioning refers to a technique for determining imagesat different depths through the thickness of an object 30 using aquantitative DIC device 100 with a wavefront sensor 110/210. Any type ofwavefront sensor 110/210 can be used. In some embodiments, polarizationeffects are ignored. For simplicity, it is assumed that in theseembodiments this technique is premised on a light field that is linearlypolarized and on no interactions in the light field depolarizing or inany other way disrupting the polarization of the light field.

The approach to the computed depth sectioning technique is primarilybased on two concepts. According to a first concept, the light field atany given plane z can be fully described by a complete set of spatiallyvarying amplitude and phase information. In other words, a light fieldat plane z can be described by Eq. (11) as:

ψ(x,y,z)=A(x,y,z)exp(iφ(x,y,z)),  (11)

where ψ(x, y, z) is the light field at plane z, A(x, y, z) is theamplitude at plane z and φ(x, y, z) is the phase at plane z. A secondconcept provides the Huygen's principle which states that the lightfield at an earlier or later (higher or lower z value) plane can becalculated from the light field at plane z. In other words, a knownfunction (f) connects according to Eq. (12):

ψ(x,y,z+Δz)=f(ψ(x,y,z),Δz),  (12)

where ψ(x, y, z+Δz) is the light field at plane (z+Δz). The function (f)is well known and studied in electromagnetic theory. For example, thisfunction f is described in Kraus, John Daniel, Fleisch, Daniel A.,Electromagnetics with Applications (5^(th) Ed), Chapters 4-16 (1999),which is herein incorporated by reference in its entirety for allpurposes. This computation assumes the absence of unknown scatteringobjects between plane z and plane (z+Δz).

These two concepts are powerful when applied to phase imaging inembodiments of the invention. It implies that if one can measure thephase and amplitude distribution at the plane of the sensor (plane z),one can calculate and render the light field distributions at differentheights (different (z+Δz) planes) above the sensor. The light fielddistributions are, in effect, images at those chosen planes. Accordingto this treatment, if a wavefront sensor can measure the two dimensionalphase and amplitude data at a light detector plane z, the quantitativeDIC device can use this data to numerically reconstruct the image andnumerically propagate it to any plane z+Δz above or below the plane z ofthe light detector.

The propagation of the light field is governed by Maxwell's equationsentirely. If one can measure the phase and amplitude distribution of thelight field at the sensor, one can take that information and calculatethe light field distribution at any given plane above the sensor (orbelow the sensor). The amplitude distribution of the computed lightfield is equivalent to the traditional microscope image taken at thefocal plane set at z+Δz. This treatment is strictly true if no unknownobject is present between the plane z and z+Δz.

FIG. 11 illustrates a schematic diagram illustrating this propagationapproach, according to embodiments of the invention. In the illustratedexample, an illumination source 20 provides light. The lightdistribution of a first wavefront 120(a) is ψ(x, y, z+Δz) at plane z+Δz.In this example, plane z refers to a plane perpendicular to the z-axisand coinciding with the surface 230(a) of the light detector 230 ofwavefront sensor 110/210. Plane z+Δz refers to a plane parallel to planez and at a distance Δz from plane z.

In FIG. 11, the light detector 230 of the wavefront sensor 110/210measures the light distribution ψ(x, y, z) of the second (detected)wavefront 120(b) at plane z. The light detector 230 measures thetwo-dimensional amplitude and two-dimensional phase gradient data in twoorthogonal directions, associated with the second (detected) wavefront120(b) at plane z based on the measured light distribution ψ(x, y, z). Aprocessor 152 of the wavefront sensor 110/210 or of the host computer150 numerically reconstructs a third (reconstructed) wavefront 120(c)having the light distribution ψ(x, y, z) using the measured phase andamplitude information of the detected wavefront 120(b). The processor152 of the wavefront sensor 110/210 or of the host computer 150calculates and renders a light distribution ψ_(calculated)(x, y, z+Δz)of a fourth (propagated) wavefront 120(d) at a plane z+Δz based on thereconstructed light distribution ψ(x, y, z). That is, the processor 152of the wavefront sensor 110/210 or of the host computer 150 numericallypropagates the reconstructed wavefront 120(c) from plane z to the planez+Δz to generate the fourth (propagated) wavefront 120(d).

The imaging of an unknown but weak scatterer (e.g., a transparentobject) can be performed computationally using the same mathematicalframe work as described above—by ignoring the presence of the scattererduring the calculation and back-computing the light field at z+Δz. Usingthis frame work by ignoring the presence of the object, the quantitativeDIC device can image an object by computationally back propagating areconstructed image of the object at plane z to parallel planes aboveand below the plane z.

FIG. 12 is a schematic diagram of the propagation approach using aprocessor, according to embodiments of the invention. In this example,the illumination source generates a uniform wavefront 120(e) associatedwith a uniform light distribution of ψ(x, y, z+Δz′) at plane z+Δz′.Plane z+Δz′ refers to a plane between the illumination source 20 and theobject 30, that is parallel to plane z, and that is at a distance Δz′from plane z. At plane z+Δz, the object 30 induces a first (induced)wavefront 120(a) associated with a light distribution of ψ(x, y, z+Δz).Plane z+Δz refers to a plane parallel to plane z and at a distance Δzfrom plane z. The light detector 230 of the wavefront sensor 110/210measures the light distribution ψ(x, y, z) of the second (detected)image wavefront 120(b) at plane z. The light detector 230 measures theamplitude and phase gradient of the second (detected) image wavefront120(b) at plane z based on the light distribution ψ(x, y, z). In thisexample, plane z refers to a plane perpendicular to the z-axis andcoinciding with the surface 230(a) of the light detector 230 ofwavefront sensor 110/210.

A processor 152 of the wavefront sensor 110/210 or of the host computer150 numerically reconstructs the light distribution ψ(x, y, z) of athird (reconstructed) image wavefront 120(c) based on the measured phaseand amplitude information of the detected image wavefront 120(b). Theprocessor 152 of the wavefront sensor 110/210 or of the host computer150 calculates and renders a computed light distributionψ_(calculated)(x, y, z+Δz) of a first (propagated) image wavefront120(d) at a plane z+Δz based on the reconstructed light distributionψ(x, y, z). That is, the processor 152 of the wavefront sensor 110/210or of the host computer 150 numerically propagates the reconstructedimage wavefront 120(c) from plane z to the plane z+Δz to generate afirst propagated image wavefront 120(d) that approximates the firstinduced wavefront 120(a). The processor 152 of the wavefront sensor110/210 or of the host computer 150 also calculates and renders acomputed light distribution ψ_(calculated)(x, y, z+Δz′) of a secondpropagated image wavefront 120(f) at a plane z+Δz′ based on thereconstructed wavefront 120(c) or based on the first propagated imagewavefront 120(d). The second propagated image wavefront 120(f)approximates the image wavefront 120(e) associated with the uniformlight distribution ψ(x, y, z+Δz). In the same way, the processor 152 ofthe wavefront sensor 110/210 or of the host computer 150 can numericallypropagate the reconstructed image wavefront 120(c) or any previouslypropagated image wavefronts to other z-planes at any height through thethickness of the object 30. The processor 152 of the wavefront sensor110/210 or of the host computer 150 can compute the depth sectioning ofthe object 30 by propagating image wavefronts at different heightsthrough the object 30.

The amplitude distribution of the computed light field is equivalent tothe traditional microscope image taken at the focal plane set at z+Δz.In fact, it can be identical. In the traditional microscope, thecalculation is performed optically by the optical elements. By adjustingthe optics (e.g., lenses), one can bring different planes into focus buteffectively what one is doing is making slight adjustments to theoptical computing process.

FIGS. 13( a) and 13(b) are schematic drawings of a focusing approachtaken by a traditional microscope. In this approach, the opticalelements (e.g., lenses) are moved to bring different planes into focus.In FIG. 13( a), the microscope is adjusting the optics to bring planez+Δz into focus. In FIG. 13( b), the microscope is adjusting the opticsto bring plane z+Δz′ into focus.

In FIG. 13( a), the illumination source generates a uniform wavefront120(e) associated with a uniform light distribution of ψ(x, y, z+Δz′) atplane z+Δz′. Plane z+Δz′ refers to a plane between the illuminationsource 20 and the object 30, that is parallel to plane z, and that is ata distance Δz′ from plane z. At plane z+Δz, the object 30 induces afirst (induced) wavefront 120(a) associated with a light distribution ofψ(x, y, z+Δz). Plane z+Δz refers to a plane parallel to plane z and at adistance Δz from plane z. The optical elements 500 are placed at adistance away from the sensor 510 to calculate a light distribution ofψ_(calculated)(x, y, z+Δz) to focus an image wavefront 120(f) on planez+Δz.

In FIG. 13( b), the illumination source generates a uniform wavefront120(e) associated with a uniform light distribution of ψ(x, y, z+Δz′) atplane z+Δz′. Plane z+Δz′ refers to a plane between the illuminationsource 20 and the object 30, that is parallel to plane z, and that is ata distance Δz′ from plane z. At plane z+Δz, the object 30 induces afirst (induced) wavefront 120(a) associated with a light distribution ofψ(x, y, z+Δz). Plane z+Δz refers to a plane parallel to plane z and at adistance Δz from plane z. The optical elements 500 are placed at adistance away from the sensor 510 to calculate a light distribution ofψ_(calculated)(x, y, z+Δz′) to focus an image wavefront 120(f) on planez+Δz′.

In some cases, the process may not be perfect because one may notachieve a good image if the scatterer is thick and/or highly scatteringbecause the assumption that the scatterer is ignorable in thecomputation process may be violated. However, this problem can affectcomputation-based depth sectioning using a quantitative DIC device andoptical-based sectioning using a traditional microscope equally. Theaxiom of ‘no free lunch’ can apply equally in both situations. For thintissue sections or cell samples, the distortion may be nominallytolerable. In practical situations, one can typically deal with a 100microns thick tissue sample before distortion starts becomingsignificant.

A sensor capable of spatially measuring amplitude and phase is requiredto compute depth sectioning of the object based on the propagationapproach. The wavefront sensors embodiments have the capability ofmeasuring the two-dimensional amplitude and phase required to computedepth sectioning of the object. In addition, the signal to noise ratioof the sensor measurements may need to be high in some cases. Otherwisethe computed images may be poor in quality.

Modifications, additions, or omissions may be made to the quantitativeDIC device 100 of any configuration or to the wavefront sensors 110 or210 of any type (e.g., SAI wavefront sensor, Shack-Hartman wavefrontsensor, and OFM wavefront sensor) without departing from the scope ofthe disclosure. The components of the quantitative DIC device 100 of anyconfiguration or to the wavefront sensors 110 or 210 of any type may beintegrated or separated according to particular needs. For example, theprocessor 152 may be a component of the light detector 230. Moreover,the operations of the quantitative DIC device 100 may be performed bymore, fewer, or other components and the operations of the wavefrontsensors 110 or 210 may be performed by more, fewer, or other components.Additionally, operations of the quantitative DIC device 100 or wavefrontsensors 110/210 may be performed using any suitable logic comprisingsoftware, hardware, other logic, or any suitable combination of thepreceding.

B. Flow Chart

FIG. 14 is a flow chart of a compute depth sectioning method using aquantitative DIC device 100 having a wavefront sensor 110/210, accordingto embodiments of the invention. The quantitative DIC device 100 used inthis method can have any suitable type of wavefront sensor 110/210.Suitable types of wavefront sensors 110/210 include a SAI wavefrontsensor, Shack-Hartmann wavefront sensor, OFM wavefront sensor, or othersuitable wavefront sensor. In addition, any suitable configuration ofwavefront sensor wavefront sensor 110/210 can be used. For example, thequantitative DIC device 100 can have a single pixel wavefront sensor110, a one dimensional array of sensor elements 210, a two-dimensionalwavefront sensor array 210, etc. If the wavefront sensor wavefrontsensor 110/210 is a single pixel wavefront sensor, a raster scanningdevice can be employed to scan the wavefront sensor or the object to geta two-dimensional reading at a single time. If the wavefront sensor110/210 is a two-dimensional wavefront sensor array, the wavefrontsensor can take a two-dimensional reading of the image wavefront at thesame time. If the wavefront sensor 110/210 is a one-dimensional array,the wavefront sensor 110/210 can read time varying data in the form ofline scans and compile the line scans to generate two-dimensionalwavefront data.

The method begins with an illumination source or sources 20 providinglight (step 600). Without the object 30 being present, the lightgenerates an initialization wavefront with a light distribution of ψ(x,y, z+Δz′) at plane z+Δz′ of the illumination source 20. The illuminationsource or sources 20 can provide light in any suitable direction(s). Ifthe quantitative DIC device 100 is a component of a camera system,multiple illumination sources 20 such as a flash, ambient light, etc.may provide light from multiple directions. If the quantitative DICdevice 100 is a component of a microscope, a single illumination source20 may provide light in a single direction along the z-axis toward thewavefront sensor. In one embodiment, a wavefront relay system 130 relaysor projects the wavefront to the wavefront sensor. In other embodiments,the wavefront relay system 130 is eliminated and the wavefront isprojected directly to the wavefront sensor 110/210.

Next, the wavefront sensor 110/210 measures the light distribution ψ(x,y, z) of the initialization wavefront 120 at plane z (step 602). Theinitialization wavefront is received through the structured apertures300 by the light detecting elements 232 of the light detector 230. Ifthe wavefront sensor is a single pixel wavefront sensor 110, the lightdetector 230 uses a raster scanning device 140 to scan the wavefrontsensor 110/210 or the object 30 to generate a reading of the twodimensional light intensity distribution at a single time. If thewavefront sensor is a two dimensional sensor array, the light detector230 can read the two dimensional light intensity distribution in asnapshot reading without raster scanning. If the wavefront sensor is aone dimensional sensor array using an OFM scheme (OFM wavefront sensor),the light detector 230 reads time varying data as the object 30 passesthrough the fluid channel. The time varying data can be in the form ofline scans which can be compiled to generate the two dimensional lightdistribution.

The processor 152 of the wavefront sensor 110/210 or of the hostcomputer 150 separates the light projection/distribution of eachstructured aperture 300 from the light projection/distribution fromother structured apertures 300 (step 604). Once separated, the lightdistributions/projections can be used to map the light detectingelements 232 of the light detector 230 to the structured apertures 300.

Any suitable technique for separating the projections/distributions canbe used. In one embodiment, separation can be performed by suppressingthe crosstalk from adjacent projections of the wavefront 120 throughadjacent structured apertures 300. In this embodiment, the processor 152can determine the maximum intensity values of the light distribution.The processor 152 can then determine the light detecting elements 232that have read the maximum intensity values. The processor 152 candetermine the midpoint on the light detector 230 between the lightdetecting elements 232 reading the maximum intensity values. Theprocessor 152 can then use the midpoint to separate the light detectingelements reading the light distribution from particular structuredapertures 300. Alternatively, a predefined number of light detectingelements 232 around each light detecting element 232 with the maximumintensity value can define the light detecting elements 232 associatedwith each structured aperture 300.

Next, the processor 152 predicts the center of eachprojection/distribution 300 (step 606). Any method can be used topredict the initial centers. In one embodiment, the processor 152 maydetermine that the center of each projection/distribution is the lightdetecting element having the highest intensity value.

The object 30 is introduced (step 608). In other embodiments, steps 602through 606 can be performed after step 614 and before step 616. Theobject 30 can be introduced using any suitable technique. For example,the object 30 may be injected with a fluid sample into an input port ofthe quantitative DIC device 100.

Once introduced, the object 30 alters the light from the illuminationsource 20 and induces an image wavefront 120 at z+Δz having a lightdistribution ψ(x, y, z+Δz). Referring to FIGS. 15( a), 15(b), and 15(c),a wavefront sensor 210 is shown with an image wavefront induced by anobject, according to an embodiment of the invention. FIG. 15( a) is aside view of components of the wavefront sensor 210, according to anembodiment of the invention. The wavefront sensor 210 includes anaperture layer 220 having an array of three structured apertures 300(a),300(b) and 300(c) along the x-axis. FIG. 15( b) is a top view ofcomponents of the wavefront sensor 210 in FIG. 15( a), according to anembodiment of the invention. FIG. 15( c) is a sectional view ofcomponents of the wavefront sensor 210 in FIG. 15( a) through the centerof structured aperture 300(c), according to an embodiment of theinvention. Although three structured apertures 300(a), 300(b) and 300(c)are shown, any number of structured apertures can be used. In addition,any type of structured aperture (e.g., four hole, single hole, etc.) maybe used.

In FIGS. 15( a), 15(b), and 15(c), the wavefront sensor 210 alsoincludes a light detector 230 having a surface 230(a) and a transparentlayer 240 between the light detector 230 and the aperture layer 220. Thetransparent layer 240 has a thickness D. Although the illustratedexample shows the light detector 230 comprising a two-dimensional arrayof light detecting elements, any suitable number or configuration oflight detecting elements 232 can be used. For example, a single lightdetecting element 232 can be used.

Returning to FIG. 14, the wavefront sensor measures the lightdistribution ψ(x, y, z) of the wavefront induced by the object 30 (step610). The wavefront is measured at plane z of the light detectorcorresponding to surface 230(a) of the light detector 230.

In the illustrated embodiment shown in FIGS. 15( a), 15(b), and 15(c),the wavefront 120 is received through the structured apertures 300(a),300(b) and 300(c) in the aperture layer 220 by the light detectingelements 232 of the light detector 230. The light detector 230 can takea two-dimensional snapshot reading of the light distribution or can usea raster scanning device 140 to scan the wavefront sensor to get atwo-dimensional reading at a single time. In another embodiment, thelight detector 230 can read time varying intensity information throughstructured apertures 300 of an OFM wavefront sensor. In this case, thelight distribution is compiled from line scans of the time varyinginformation.

Returning to FIG. 14, the processor 152 of the wavefront sensor or thehost computer separates the light distribution/projection (step 612). Inthe illustrated embodiment shown in FIGS. 15( a), 15(b), and 15(c), theprocessor 152 separates the light distributions 800(a), 800(b) and800(c) from a particular light transmissive region 232 of a structuredaperture 300 from the light distribution projected from lighttransmissive regions 232 of other structured apertures 300. For example,the processor 152 separates the light distribution 800(a) associatedwith structured aperture 300(a) from the light distributions 800(b) and800(c) associated with the structured apertures 300(b) and 300(c). Theseparation of the light distributions/projections can be used to map thelight detecting elements 232 of the light detector 230 to the lighttransmissive region(s) of the structured apertures 300.

Any suitable method of separating the projections/distributions 800(a),800(b) and 800(c) can be used. In one embodiment, separation can beperformed by suppressing the crosstalk from adjacent projections of thewavefront 120 through adjacent structured apertures 300(a), 300(b) and300(c). The processor 152 of the quantitative DIC device 100 determinesthe maximum intensity values 820(a), 820(b), 820(c), and 820(d) of thelight distributions 800(a), 800(b) and 800(c) in both orthogonal x- andy-directions. The processor 152 then determines the light detectingelements 232 reading the maximum intensity values 820(a), 820(b) and820(c). The processor 152 can determine the midpoint on the lightdetector 230 between the light detecting elements 232 reading themaximum intensity values 820(a), 820(b), 820(c), and 820(d). Theprocessor 152 can then use the midpoint to separate the light detectingelements reading the light distribution from particular structuredapertures 300. Alternatively, a predefined number of light detectingelements 232 around the light detecting element 232 reading the maximumintensity value can define the light detecting elements 232 associatedwith each structured aperture 300. For example, all light detectingelements 232 within three light detecting elements of the one readingthe maximum intensity value can be associated with the lightdistribution from a particular structured aperture 300.

Returning to FIG. 14, the processor 152 also predicts the center of theprojections associated with the structured apertures (step 614). Anymethod can be used to predict the centers. In one embodiment, theprocessor 152 may determine that the center of each separatedprojection/distribution is the light detecting element having thehighest intensity value.

Next, the processor 152 determines the x and y offsets (step 616). Theprocessor 152 can determine the offsets from the change in position ofthe center in both the x and y directions of each projection before andafter an object is introduced. In FIGS. 15( a), 15(b), and 15(c), theprocessor 152 determines the offset 810(a) at structured aperture 300(a)in the x-direction, offset 810(b) at structured aperture 300(b) in thex-direction and offset 810(c) at structured aperture 300(c) in thex-direction. The processor 152 also determines an offset 810(d) ataperture 300(c) in the y-direction. Although not shown, the processor152 also determines offsets in the y-direction at apertures 300(a) and300(b). In another embodiment, the processor 152 may determine theoffsets from a change in the position of another portion of eachprojection before and after the object is introduced.

Returning to FIG. 14, the processor 152 determines the phase gradient intwo orthogonal directions (step 618). The processor 152 can determinethe local phase gradient in two orthogonal directions based on theoffsets in the x and y directions using Eqns. (2) and (3). Generally,wavefront sensors having structured apertures 300 (e.g., four holeaperture or single hole aperture sensors) of some embodiments canmeasure the spatial phase gradient of the light field. Mathematically,the wavefront sensors of some embodiments measure:

G _(x)(x,y)=k _(x)(x,y)/k ₀≈(dφ(x,y)/dx)/k ₀,  (6)

G _(y)(x,y)=k _(y)(x,y)/k ₀≈(dφ(x,y)/dy)/k ₀,and  (7)

A(x,y),  (8)

where G_(x)(x, y) is the two-dimensional phase gradient in thex-direction, G_(y)(x, y) is the two-dimensional phase gradient in they-direction, and A(x, y) is the two-dimensional amplitude of thedetected wavefront. To determine the phase gradient in two orthogonaldirections based on the offsets, the processor 152 can measure the netwavefront gradient G_(x)(x, y) and G_(y)(x, y) at each aperturerespectively based on Eq. (10):

$\begin{matrix}{{{G_{x}\left( {x,y} \right)} = \frac{1}{\sqrt{1 + \left( \frac{D}{{offset}_{x}\left( {x,y} \right)} \right)^{2}}}}{and}{{{G_{y}\left( {x,y} \right)} = \frac{1}{\sqrt{1 + \left( \frac{D}{{offset}_{y}\left( {x,y} \right)} \right)^{2}}}},}} & (10)\end{matrix}$

where D is the transparent layer (spacer) thickness, the offset in thex-direction is offset_(x)(x,y) and the offset in the y-direction isoffset_(y)(x,y).

The processor 152 also measures the amplitude of the image wavefront 120(step 620). The processor 152 measures the amplitude by summing up allthe intensity values in each separated projection/distributionassociated with each structured aperture 300. With the amplitude andphase gradient information, the quantitative DIC device 100 hassufficient data to reconstruct the image wavefront at the plane z at thelight detector 230.

The quantitative DIC device 100 can mathematically reconstruct (unwrap)the detected wavefront by combining the measured data appropriatelyusing an unwrapping method (step 622). One unwrapping method is given byEq. (9):

$\begin{matrix}{{\psi_{measured}\left( {x,y,z} \right)} = {{A_{measured}\left( {x,y,z} \right)}{{\exp \left( {\; {k_{0}\begin{pmatrix}{\int_{0}^{x}{G_{x}\left( {{\left( {x,{y = 0}} \right){x}} +} \right.}} \\{\int_{0}^{y}{{G_{y}\left( {{x = x},y} \right)}{y}}}\end{pmatrix}}} \right)}.}}} & (9)\end{matrix}$

Numerous approaches for reconstructing a field distribution exist(unwrapping). Some examples of suitable unwrapping methods include theAffine transformation method, the least squares method, the FrankotChellappa methods of wrapping, etc. The unwrapping methods should allreturn the same answer if the signal to noise ratio (SNR) of themeasurements approaches infinity. The unwrapping methods may vary intheir performance based on the quantity and type of noise present in themeasurements.

The processor 152 propagates the reconstructed wavefront at plane z toone or more planes z+Δz intersecting the object 30 (step 624). Theprocessor 152 propagates the reconstructed wavefront having adistribution ψ(x, y, z) to the other planes based on Eq. (12): ψ(x, y,z+Δz)=f(ψ(x, y, z), Δz) where ψ(x, y, z+Δz) is the light distribution atplane (z+Δz) intersecting the object 30. The function (f) is well knownand studied in electromagnetic theory. For example, this function f isdescribed in Kraus, John Daniel, Fleisch, Daniel A., Electromagneticswith Applications (5^(th) Ed), Chapters 4-16 (1999).

The processor 152 may propagate the reconstructed wavefront to anynumber of planes (z+Δz) at different depths through the object 30. Inone embodiment, the object 30 may have a dimension h along the z axisand may be located adjacent to the surface 230(a) of the light detector230. The processor 152 may propagate the reconstructed wavefront n times(e.g., 100, 1000, 2000, etc.) through the planes z+Δz_(k=1 ton) whereΔz_(k)=Δz₁+hk/n. In this case, the reconstructed wavefront is propagatedto n depths starting at the surface 230(a) where n=1 and incrementallyincreasing the depths by hk/n. In other embodiments, particular depthsare used to image a region of the object 30. For example, the processor152 may propagate the reconstructed wavefront to a plane at the middleof the object 30.

The processor 152 generates two-dimensional and three-dimensional images(step 626). In one embodiment, the processor 152 generatestwo-dimensional images based on the reconstructed wavefront, theintensity distribution, the phase gradient distribution in thex-direction, and/or the phase gradient distribution in the y-direction.The processor 152 can also combine the two-dimensional images togenerate a three-dimensional image of the object 30 or portions of theobject 30.

In some embodiments, an output device (e.g., a printer, display, etc.)of the quantitative DIC device 100 can output various forms of data. Forexample, the quantitative DIC device 100 can output a two-dimensionallocal intensity image map, a two-dimensional phase gradient image map inand x-direction, a two-dimensional phase gradient image map in andy-direction, two-dimensional reconstructed image, a two-dimensionalpropagated image, and/or a three-dimensional image. The quantitative DICdevice can also further analyze the two-dimensional wavefront data. Forexample, the quantitative DIC device 100 can use the intensity data toanalyze biological properties of the object 30.

Modifications, additions, or omissions may be made to the method withoutdeparting from the scope of the disclosure. The method may include more,fewer, or other steps. Additionally, steps may be performed in anysuitable order without departing from the scope of the disclosure.

The quantitative DIC device 100 of some embodiments can be used invarious applications such as biological sample imaging. FIG. 16( a) isan intensity/amplitude image taken of a starfish embryo using aquantitative DIC microscope having an SAI wavefront sensor, according toan embodiment of the invention. FIG. 16( b) is an image based on phasegradient in the x direction taken of a starfish embryo using aquantitative DIC device having an SAI wavefront sensor, according to anembodiment of the invention. FIG. 16( c) is an image based on phasegradient in the y direction taken of a starfish embryo using aquantitative DIC device having an SAI wavefront sensor, according to anembodiment of the invention. FIGS. 16( d), 16(e) and 16(f) showcase someof the unwrapping algorithms when applied to the raw amplitude,differential phase x and differential phase y data of FIGS. 16( a),16(b) and 16(c), according to embodiments of the invention. FIG. 16( d)is an image reconstructed using the least squares unwrapping methodapplied to the raw amplitude/intensity, phase gradient in thex-direction and phase gradient in the y-direction shown in FIGS. 16( a),16(b) and 16(c) respectively. FIG. 16( e) is an image reconstructedusing the Frankot Chellappa unwrapping method applied to the rawamplitude/intensity, phase gradient in the x-direction and phasegradient in the y-direction shown in FIGS. 16( a), 16(b) and 16(c)respectively. FIG. 16( f) is an image reconstructed using the Affinetransformation unwrapping method applied to the raw amplitude/intensity,phase gradient in the x-direction and phase gradient in the y-directionshown in FIGS. 16( a), 16(b) and 16(c) respectively. With the wavefrontcomputed (reconstructed), we can then perform computation-basedsectioning via the approach outlined in the first section.

FIG. 17( a) is an image of potato starch storage granules in immersionoil taken by a conventional transmission microscope. FIG. 17( b) is animage of potato starch storage granules in immersion oil taken by aconventional DIC microscope. FIG. 17( c) is an intensity image of potatostarch storage granules in immersion oil taken by a quantitative DICdevice in a microscope system, according to an embodiment of theinvention. FIG. 17( d) is an artifact-free x-direction phase image ofpotato starch storage granules in immersion oil taken by a quantitativeDIC device in a microscope system, according to an embodiment of theinvention. FIG. 17( e) is an artifact-free y-direction phase image ofpotato starch storage granules in immersion oil taken by a quantitativeDIC device 100 in a microscope system, according to an embodiment of theinvention.

Birefringent objects such as the potato starch storage granules in FIGS.17( a)-17(e) can alter polarization of the two displaced fields in aconventional DIC microscope, such that the subsequent combination of thetwo files in FIG. 2 is no longer describable by Eq. (1). This can giverise to the Maltese-cross-like pattern artifacts in the resultingconventional DIC images. FIG. 17( b) shows Maltese-cross-like patternartifacts in the conventional DIC image of the potato starch storagegranules.

In some embodiments, a quantitative DIC device 100 uses unpolarizedlight and does not rely on polarization for image processing. In theseexamples, the quantitative DIC device 100 can image birefringent samples(e.g., potato starch storage granules) without artifacts. FIGS. 17( d)and 17(e) show images of birefringent samples taken by a quantitativeDIC device in a microscope system. In these figures, the birefringentimages of the potato starch storage granules i.e. birefringent samplesare without artifacts. Also, the dark absorption spots of the potatostarch granules in the center of the intensity images in FIG. 17( c) donot appear in the phase images in FIGS. 17( d) and 17(e). This indicatesthat the quantitative DIC device 100 can separate the intensityvariations of the image wavefront from the phase variations. This isadvantageous over conventional DIC devices which cannot distinguishbetween the effects of absorption and phase variation and cannot providequantitative phase measurements.

In one embodiment, the SAI wavefront sensor, a Shack-Hartmann wavefrontsensor, or an OFM wavefront sensor can be placed into a camera system.One advantage to placing the wavefront sensor into a camera is that thecamera can detect the phase gradient of the projected object wavefrontin addition to the intensity information about the projected objectwavefront.

In some embodiments, wavefront sensors 110/210 can be used withbroadband illumination and/or monochromatic illumination. Wavefrontsensors 110/210 apply to a monochromatic light field distribution inwhich k is well defined at each point on the image plane. However,wavefront sensing can also be used for a broadband light source and withsituations where k at any given point may be a mix of different wavevectors. In this regard, wavefront sensors 110/210 can be used withbroadband light illumination, monochromatic illumination with mixed k,and broadband light illumination with mixed k. An example of usingbroadband illumination by wavefront sensors 110/210 can be found in Cui,Xiquan, Lew, Matthew, Yang, Changhuei, Quantitative differentialinterference contrast microscopy based on structured-apertureinterference,” Applied Physics Letters Vol. 93 (9), 091113 (2008), whichis hereby incorporated by reference in its entirety for all purposes.

In one embodiment, the diffraction spot size in a SAI wavefront sensorand the focal spot size in the Shack-Hartmann wavefront sensor of aquantitative DIC device 100 can be used to determine the spread of wavevector k at any given image point. The quantitative DIC device 100 inthis embodiment can render images where the extent of scattering isplotted.

In one embodiment, the quantitative DIC device 100 can determine theproportionality of the phase gradient response of the wavefront sensor110/210. The quantitative DIC device 100 measures the interferencepattern as the wavefront sensor 110/210 is illuminated by a suitableillumination source (e.g., a collimated He—Ne laser beam) with a lighthaving suitable properties (e.g., 632.8 nm wavelength, 25 mm beamdiameter, and 4 mW power) and with a range of incident angles. The totaltransmission and the offsets of the zero-order spot in both x and ydirections can be computed with a suitable method such as a least-square2D Gaussian fit. The relationship between the offsets of the zero orderspot and the normalized phase gradient can be approximately linear. Thequantitative DIC device 100 estimates the constant that represents theapproximately linear relationship between the offsets of the zero orderspot and the normalized phase gradient.

V. Computer Devices

FIG. 18 shows a block diagram of subsystems that may be present incomputer devices that are used in quantitative DIC device 100, accordingto embodiments of the invention. For example, the host computer 150 orwavefront sensor 110/210 may use any suitable combination of componentsin FIG. 18.

The various components previously described in the Figures may operateusing one or more computer devices to facilitate the functions describedherein. Any of the elements in the Figures may use any suitable numberof subsystems to facilitate the functions described herein. Examples ofsuch subsystems or components are shown in a FIG. 18. The subsystemsshown in FIG. 18 are interconnected via a system bus 775. Additionalsubsystems such as a printer 774, keyboard 778, fixed disk 779 (or othermemory comprising computer readable media), monitor 776, which iscoupled to display adapter 782, and others are shown. Peripherals andinput/output (I/O) devices, which couple to I/O controller 771, can beconnected to the computer system by any number of means known in theart, such as serial port 777. For example, serial port 777 or externalinterface 781 can be used to connect the computer apparatus to a widearea network such as the Internet, a mouse input device, or a scanner.The interconnection via system bus allows the central processor 152 tocommunicate with each subsystem and to control the execution ofinstructions from system memory 772 or the fixed disk 779, as well asthe exchange of information between subsystems. The system memory 772and/or the fixed disk 779 may embody a computer readable medium. Any ofthese elements may be present in the previously described features. Acomputer readable medium according to an embodiment of the invention maycomprise code for performing any of the functions described above.

In some embodiments, an output device (e.g., the printer 774) of thequantitative DIC device 100 can output various forms of data. Forexample, the quantitative DIC device 100 can output a two-dimensionallocal intensity image map, a two-dimensional phase gradient image map inand x-direction, a two-dimensional phase gradient image map in andy-direction, two-dimensional reconstructed image, a two-dimensionalpropagated image, and/or a three-dimensional image. The quantitative DICdevice 100 can also further analyze the two-dimensional wavefront data.For example, the quantitative DIC device 100 can use the intensity datato analyze biological properties of the object 30.

It should be understood that the present invention as described abovecan be implemented in the form of control logic using computer softwarein a modular or integrated manner. Based on the disclosure and teachingsprovided herein, a person of ordinary skill in the art will know andappreciate other ways and/or methods to implement the present inventionusing hardware and a combination of hardware and software.

Any of the software components or functions described in thisapplication, may be implemented as software code to be executed by aprocessor using any suitable computer language such as, for example,Java, C++ or Perl using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructions,or commands on a computer readable medium, such as a random accessmemory (RAM), a read only memory (ROM), a magnetic medium such as ahard-drive or a floppy disk, or an optical medium such as a CD-ROM. Anysuch computer readable medium may reside on or within a singlecomputational apparatus, and may be present on or within differentcomputational apparatuses within a system or network.

A recitation of “a”, “an” or “the” is intended to mean “one or more”unless specifically indicated to the contrary.

The above description is illustrative and is not restrictive. Manyvariations of the disclosure will become apparent to those skilled inthe art upon review of the disclosure. The scope of the disclosureshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to thepending claims along with their full scope or equivalents.

One or more features from any embodiment may be combined with one ormore features of any other embodiment without departing from the scopeof the disclosure. Further, modifications, additions, or omissions maybe made to any embodiment without departing from the scope of thedisclosure. The components of any embodiment may be integrated orseparated according to particular needs without departing from the scopeof the disclosure.

All patents, patent applications, publications, and descriptionsmentioned above are herein incorporated by reference in their entiretyfor all purposes. None is admitted to be prior art.

1. A method for computing depth sectioning of an object using aquantitative differential interference contrast device having awavefront sensor with one or more structured apertures, a light detectorand a transparent layer between the one or more structured apertures andthe light detector, the method comprising: receiving light by the lightdetector through the one or more structured apertures; measuring anamplitude of an image wavefront based on the received light; measuring aphase gradient in two orthogonal directions of the image wavefront basedon the received light; reconstructing, by a processor, the imagewavefront using the measured amplitude and phase gradient; andpropagating, by the processor, the reconstructed wavefront to a firstplane intersecting an object at a first depth.
 2. The method of claim 1,further comprising propagating, by the processor, the reconstructedwavefront to a second plane intersecting the object at a second depth.3. The method of claim 2, further comprising generating athree-dimensional image of a portion of the object based on thepropagated wavefronts at the first and second planes.
 4. The method ofclaim 1, further comprising: propagating, by the processor, thereconstructed wavefront to a plurality of parallel planes intersectingthe object, the planes substantially parallel to a surface of the lightdetector and at depths substantially extending through a dimension ofthe object; and generating a three-dimensional image of the object basedon the propagated wavefronts at the first plane and the plurality ofplanes.
 5. The method of claim 1, generating a two-dimensional image ofthe object at the first depth based on the propagated wavefront at thefirst plane.
 6. The method of claim 5, wherein the two-dimensional imageis a cross-sectional image of the object.
 7. The method of claim 1,wherein measuring an amplitude of the image wavefront based on thereceived light comprises: separating intensity data of the receivedlight associated with each structured aperture; measuring a localamplitude associated with each structured aperture by summing theseparated intensity data associated with each structured aperture; andgenerating the amplitude of the image wavefront by compiling the localamplitudes measured at each structured aperture.
 8. The method of claim1, wherein measuring a phase gradient in two orthogonal directions ofthe image wavefront based on the received light comprises: separatingintensity data of the received light associated with each structuredaperture; measuring offsets in two orthogonal directions of the receivedlight at each structured aperture before and after the object isintroduced; and measuring a local phase gradient in two orthogonaldirections at each structured aperture based on the offsets; andgenerating the phase gradient in two orthogonal directions by compilingthe local phase gradients measured at each structured aperture.
 9. Themethod of claim 1, further comprising introducing the object whichalters light from one or more illumination sources.
 10. The method ofclaim 1, further comprising focusing the light to a focal point on thelight detector with a microlens within a structured aperture of thearray of structured apertures.
 11. The method of claim 1, whereinreceiving light by the light detector through the one or more structuredapertures comprises: generating, by the light detector, time varyingdata associated with the light received through the one or morestructured apertures as the object moves through a fluid channel; andcompiling the time varying data to generate a two-dimensional lightdistribution.
 12. A wavefront sensor comprising: an aperture layerhaving one or more structured apertures; a light detector configured tomeasure the amplitude of a wavefront, and to separately measure thephase gradient in two orthogonal directions of the wavefront based onlight received through the structured apertures; and a transparent layerbetween the aperture layer and the light detector.
 13. The wavefrontsensor of claim 12, wherein the light is unpolarized.
 14. The wavefrontsensor of claim 12, further comprising a microlens in at least one ofthe structured apertures.
 15. A quantitative differential interferencecontrast device comprising: a wavefront sensor comprising an aperturelayer having one or more structured apertures, a light detectorconfigured to measure the amplitude of a wavefront, and to measure thephase gradient in two orthogonal directions of the wavefront based onlight received through the structured apertures, and a transparent layerbetween the aperture layer and the light detector; and a processorcommunicatively coupled to the wavefront sensor and configured toreconstruct the wavefront using the measured amplitude and phasegradient.
 16. The quantitative differential interference contrast deviceof claim 15, wherein the processor is further configured to propagatethe reconstructed wavefront to a first plane intersecting an object at afirst depth.
 17. The quantitative differential interference contrastdevice of claim 15, further comprising a relay system for relaying thewavefront to the wavefront sensor.
 18. The quantitative differentialinterference contrast device of claim 15, further comprising a rasterscanning device for scanning the wavefront sensor.
 19. The quantitativedifferential interference contrast device of claim 15, furthercomprising one or more illumination sources for providing light.